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18955 Rev A_ViewRNA ISH Tissue2-Plex Assay UM.book

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1. 27 Poor Gell Morphology 3523 ie a e e at at d P Ed ce de a P ta She dee 27 High Non Specific Binding on Glass Slide 27 ViewRNA ISH Tissue 2 Plex Assay User Manual Appendix A Appendix B Appendix C Appendix D Appendix E Pink Non Specific Background Where Paraffin Was 28 Hydrophobic Barrier Falls Off 28 Fast Red Signal for TYPE 1 Target is Weak or Different in 2 Plex Versus 1 Plex 28 TYPE 1 Target Signals Observed in the Channel for TYPE 6 Target 29 Co localized Fast Blue and Fast Red Signals When Using Only TYPE 6 ProbeSet 29 Sample Pretreatment Optimization Procedures 31 About Pretreatment Optimization 31 Sample Pretreatment Optimization Setup 31 Sample Preparation and Target Probe Hybridization 32 Sample Pretreatment Lookup Table 35 Evaluating Results 22443405 cae 37 Assessing Pretreatment Conditions 37 Analyzing Target Expression 38 Modified Protocols for a 1 Plex Assay 22 4 2 39 Using Frozen Tissues with ViewRNA ISH Tissue 2 Plex Assay 41 ABOUT RIS extiende eta ri Lei
2. Action 3 Protease Digestion and Fixation 30 50 min A Prepare the working protease solution using the table below as a guide Dilute the Protease QF 1 100 in prewarmed 1X PBS and briefly vortex to mix Scale reagents according to the number of assays to be run Include one slide volume overage Working Protease Solution per Slide Reagent Volume Protease QF 4 uL 1X PBS prewarmed to 40 C 396 uL Total volume 400 uL Leave slide 1 on the lab bench as it is excluded from this step Begin by removing slides 4 and 8 and flicking each to remove excess 1X PBS Tap the slide on its edge then wipe the backside on a laboratory wipe Leave remaining slides in 1X PBS Place slides 4 and 8 face up on a flat elevated platform e g an Eppendorf tube rack for ease of handling and immediately add 400 uL of the working protease solution onto the tissue section It may be necessary to spread the solution with a pipette tip Transfer the slides to the hybridization system and incubate at 40 C for 20 min After 19 min remove slides 3 6 7 and 10 from the clear staining dish and flick off excess 1X PBS Without completely drying out the sections tap the slides on their edges and then wipe the backsides on a laboratory wipe Place slides 3 6 7 and 10 face up on a flat elevated platform and immediately add 400 uL of the working protease solution onto the tissue section Transfer the slides to the
3. affymetrn User Manual ViewRNA ISH Tissue 2 Plex Assay For research use only Not for use in diagnostic procedures Trademarks Affymetrix and 8 affymetnx are trademarks of Affymetrix Inc All other trademarks are the property of their respective owners Limited License Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products Affymetrix grants you a non exclusive non transferable non sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix You understand and agree that except as expressly set forth in the Affymetrix terms and conditions no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product In particular no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided licensed or specifically recommended by Affymetrix for such use Citing ViewRNA in Publications When describing a procedure for publication using this product please refer to it as the ViewRNA ISH Tissue 2 Plex Assay Disclaimer Affymetrix Inc reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure accuracy Af
4. Avoid any mounting medium containing alcohol or any cover slipping method requiring alcohol dehydration Endogenous Alkaline Phosphatase Activity Table 4 5 Troubleshooting Endogenous Alkaline Phosphatase Activity Probable Cause Recommended Action Endogenous alkaline phosphatase activity Verify alkaline phosphatase activity by incubating protease treated sample with Fast Red Substrate or Fast Blue Substrate If endogenous AP activity is present diffused signals which can be weak or strong will appear Inactivate endogenous AP with 0 2 M HCI at RT for 10 min before the protease step Wash samples twice with 1X PBS before proceeding to protease digestion Chapter 4 Troubleshooting 27 Tissue Detachment From Slide Table 4 6 Troubleshooting Tissue Detachment From Slide Probable Cause Recommended Action Improper tissue preparation Make sure that the tissue preparation is as recommended in Tissue Preparation Guidelines on page 7 including fixation time and reagent thickness of sections brand of positively charged glass slide and baking of the sections at 60 C for 1 hr before storing at 20 C Insufficient baking of slides Verify that the 60 min at 60 C baking step was performed prior to storage of slides at 20 C and again just before the deparaffinization step to ensure adhesion of tissue to slide Incorrect pretreatment conditions Perform full pretreatment optimization pro
5. Rat Brain spinal cord Important Procedural Notes This protocol requires overnight fixation in chilled 4 NBF prior to starting Day 1 of the assay See Step 2 Fix Tissue Overnight on page 42 Samples should be freshly sectioned at 12 1 um and mounted onto one of the following positively charged glass slides a Leica Non Clipped X tra Slides 1 mm white P N 3800200 in US Canada and Asia Pacific regions or P N 3800210 in Europe n Fisherbrand Superfrost Plus Slides white label Fisher Scientific Cati 12 550 15 avoid other colored labels as they tend to give high background Prepared frozen tissue slides should be used immediately in the assay or can be stored at 80 C for up to 6 months Perform optimization for Protease only see Table A 1 on page 31 for recommended times No heat treatment step required 42 ViewRNA ISH Tissue 2 Plex Assay User Manual Modifications to Part 1 Sample Preparation and Target Probe Hybridization The following procedural steps replace Step 1 to Step 5 in Part 1 Sample Preparation and Target Probe Hybridization on page 14 Table E 1 ViewRNA ISH Tissue 2 Plex Assay Sample Preparation and Target Probe Hybridization for Frozen Tissues Step Action 1 Prepare and Chill Add 178 mL 1X PBS and 22 mL 37 formaldehyde to a 200 mL capacity container Mix well and chill 10 NBF on ice for 1 hr 2 FixTissue Pour chilled 1096 NBF into a clear staining di
6. control slide Working Probe Set Solution per Slide Reagent Volume Probe Set Diluent QT prewarmed to 40 C 380 uL ViewRNA TYPE 1 Probe Set 10 uL ViewRNA TYPE 6 Probe Set 10 uL Total volume 400 uL B Remove each slide and flick it to remove excess 1X PBS Without completely drying out the sections tap the slides on the edge and then wipe the backside on a laboratory wipe C Place the slides face up on a flat elevated platform and immediately add 400 uL of prewarmed Probe Set Diluent QT to the negative probe control and 400 uL of working probe set solution to each test sample D Transfer the slides to the hybridization system and incubate at 40 C for 2 hr Wash Slides 8 min Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer B After incubation decant the working probe set solution from the slides and insert them into the slide rack C Wash the slides 3 times each time with 200 mL of fresh Wash Buffer at RT for 2 min with constant and vigorous agitation Click for a helpful video D Ifyou plan to perform the assay over the course of two days go to Step 9 Optional Stop Point Otherwise proceed to Step 12 PreAmplifier Hybridization on page 18 to complete the entire assay in one day Optional Stop Point 1 minute A Store slides in a clear staining dish containing 200 mL of Storage Buffer at RT for up to 24 hr Cover the dish with a lid or sea
7. 07J91 010 110V 07J68 001 Tissue culture incubator with gt 85 humidity and 0 CO and 3 aluminum slide racks for transferring slides to incubator during Major laboratory hybridization supplier ViewRNA Temperature Validation Kit Affymetrix Water proof remote probe thermometers validated for 90 100 C VWR Fume hood Major laboratory supplier Isotemp hot plates Fisher Scientific 11 300 49SHP 120V 11 302 49SHP 230V Table top microtube centrifuge Major laboratory supplier Water bath capable of maintaining 40 1 C Major laboratory supplier Vortexer Major laboratory supplier Dry incubator or oven capable of maintaining 60 C for baking slides Affymetrix or equivalent QS0704 120V or QS0714 220V Microplate shaker optional for washing steps VWR or equivalent Microscope and imaging equipment See Table 1 7 on page 6 6 ViewRNA ISH Tissue 2 Plex Assay User Manual Microscopy and Imaging Equipment Guidelines The stains used to label RNA in the ViewRNA ISH Tissue 2 Plex Assay can be visualized using brightfield or fluorescence microscopy Table 1 6 Stains for ViewRNA ISH Tissue 2 Plex Assay Detect Staining Reagent Stain Color Brightfield View Fluorescence View Table 1 7 ViewRNA ISH Tissue 2 Plex Assay Imaging Options RNA 1 using TYPE 1 probe Fast Red Red Red RNA 2 using TYPE 6 probe Fas
8. Mix TYPE 6 TYPE 1 Wey TYPE 1 Probe Set TYPE 6 Tr Amplifier Mix YPEI Probe Set TYPE 6 Visualize using brightfield 10 or fluorescence microscope Fix cells Incubate Label Probe 1 AP and permeabilize Label Probe 6 AP Fast Red Substrate 27 Zz Fast Blue Substrate Q NAH RNA 2 Sequential hybridizations 2 ViewRNA ISH Tissue 2 Plex Assay User Manual Sample Preparation FFPE tissue sections are deparaffinized and pretreated to allow unmasking of RNA and probe accessibility Target Hybridization Target specific probe pairs indicated by ZZ and ZZ in Figure 1 1 hybridize to the target RNA A typical mRNA probe set contains 20 oligonucleotide pairs For simplicity only one pair per mRNA target is shown in Figure 1 1 TYPE 1 and TYPE 6 probe sets are designed to generate red and blue signals respectively These separate yet compatible signal amplification systems provide the assay with multiplex capability Signal Amplification and Detection Signal amplification is achieved via a series of sequential hybridization steps PreAmplifiers hybridize to their respective pair of bound probe set oligonucleotides then multiple amplifiers hybridize to their respective preamplifier Next TYPE specific label probe oligonucleotides conjugated to alkaline phosphatase are sequentially hybridized to their corresponding amplifier molecules to provide up to 3 000 fold amplification
9. as bacterial dapB or sense strand of the target in order to confidently differentiate between low expressing targets and non specific background dots The ViewRNA ISH Tissue 2 Plex Assay typically has an average background of lt 1 dot 10 cells Consequently as long as your target is consistently showing an expression level above the Negative Control threshold even if the RNA target expression is extremely low e g 1 dot every 2 cells you can trust that the detection is reliably real Modified Protocols for a 1 Plex Assay This appendix provides modified and shortened assay procedures for performing a 1 plex assay using the ViewRNA ISH Tissue 2 Plex Assay Kit Whether your preference for target detection is Fast Red or Fast Blue both TYPE 1 and TYPE 6 probe sets can be used Table D 1 Modified 1 Plex Protocol for Fast Red Detection Using TYPE 1 or TYPE 6 Probe Sets Probe Set Modified Protocol Designation TYPE 1 A Perform the assay as directed through Step 15 Wash Slides on page 18 B Omit Step 16 Label Probe 6 AP Hybridization to Step 20 Quench Label Probe 6 AP on page 19 C Continue with Step 21 Label Probe 1 AP Hybridization to Step 25 Mount and Image on page 21 TYPE 6 A Perform the assay as directed through Step 17 Wash Slides on page 19 B Omit Step 18 Apply Fast Blue Substrate to Step 22 Wash Slides on page 20 C Continue with Step 23 Apply Fast Red Substrate to Step 25 Mount and Image on page 21 Table
10. clear staining dish containing 200 mL of 1X PBS C After incubation decant the AP Stop Buffer from the slides and insert them into the slide rack D Wash the slides twice each time in 200 mL of fresh 1X PBS at RT for 1 min with frequent agitation E Replace the 1X PBS with 200 mL of fresh Wash Buffer and rinse any residual PBS from the slides by moving the slide rack up and down for 1 min 20 ViewRNA ISH Tissue 2 Plex Assay User Manual Table 3 2 ViewRNA ISH Tissue 2 Plex Assay Signal Amplification and Detection Continued Step Action 21 Label Probe 1 AP A Briefly vortex and spin down Label Probe 1 AP before using Hybridization B Prepare the working Label Probe 1 AP solution using the table below as a guide Dilute Label 20 min Probe 1 AP 1 1000 in prewarmed Label Probe Diluent QF and briefly vortex to mix Scale reagents according to the number of assays to be run and include one slide volume overage Working Label Probe 1 AP Solution per Slide Reagent Volume Label Probe Diluent QF prewarmed to 40 C 399 6 uL Label Probe 1 AP 0 4 uL Total volume 400 uL C Remove each slide and flick to remove the Wash Buffer Without completely drying out the sections tap the slide on its edge and then wipe the backside on a laboratory wipe Place the slides face up on a flat elevated platform and immediately add 400 uL of working Label Probe AP solution to each tissue section D Transfer the sli
11. for 3 min with constant and vigorous agitation 18 Apply Fast Blue A Prepare the Fast Blue Substrate Add 5 mL of Blue Buffer and 105 uL of Blue Reagent 1 to a Substrate 15 mL conical tube and vortex Add 105 uL of Blue Reagent 2 and vortex Add 105 uL Blue 35 mi Reagent 3 and briefly vortex Protect from light by by wrapping in aluminum foil until use min B Remove each slide and flick it to remove the Wash Buffer Without completely drying out the sections tap the slide on its edge and then wipe the backside on a laboratory wipe Place the slides face up on a flat elevated platform and immediately add 400 pL of Fast Blue Substrate C Transfer the slides to the hybridization system and incubate in the dark at RT for 30 min 19 Wash Slides A Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer 12 min B After incubation decant the working Fast Blue Substrate from the slides and insert them into the slide rack C Wash the slides 3 times each time with 200 mL of fresh Wash Buffer at RT for 3 min with constant and vigorous agitation 20 Quench Label A Remove each slide and flick it to remove the Wash Buffer Without completely drying out the Probe 6 AP sections tap the slide on its edge and then wipe the backside on a laboratory wipe Place the 35 mi slides face up on a flat elevated platform and immediately add 400 uL of AP Stop QT min Incubate in the dark at RT for 30 min B Insert an empty slide rack into a
12. hybridization system and incubate at 40 C for 10 min After 9 min remove slides 2 5 and 9 from the clear staining dish and flick off excess 1X PBS Without completely drying out the sections tap the slides on their edges and then wipe the backsides on a laboratory wipe Place slides 2 5 and 9 face up on a flat elevated platform and immediately add 400 uL of the working protease solution onto the tissue section Transfer the slides to the hybridization system and incubate at 40 C for 10 min Pour 200 mL of 1X PBS into a clear staining dish and insert an empty slide rack into it At the end of 10 min 40 min total of incubation time decant the working protease solution from the slides insert the slides into the rack and wash gently but thoroughly by moving the rack up and down for 1 min Repeat the wash one more time with another 200 mL of fresh 1X PBS before adding slide 1 to the rack Transfer the slide rack containing all 10 slides to a clear staining dish containing 200 mL of 1096 NBF and fix at RT for 5 min under a fume hood Wash the slides twice each time with 200 mL of fresh 1X PBS for 1 min with frequent agitation Proceed to Step 7 Target Probe Set Hybridization on page 17 to continue the assay procedure 34 ViewRNA ISH Tissue 2 Plex Assay User Manual Sample Pretreatment Lookup Table Table B 1 shows a list tissues that were prepared according to the guidelines outlined in this manual Tissue Prep
13. per target RNA Visualization Sequential hybridization of TYPE 6 label probe followed by addition of Fast Blue substrate and TYPE 1 label probe followed by addition of Fast Red substrate produces blue and red precipitates dots respectively The target mRNAs are visualized using a standard brightfield and or fluorescent microscope Performance Highlights Table 1 1 Performance Highlights Specification Description Sample types Formalin fixed paraffin embedded FFPE tissue section or microarray OCT embedded frozen tissue sections Sensitivity Single RNA molecule one dot one RNA molecule Plex Detection of two target RNAs Detection Chromogenic and fluorescence Nuclear stain Hematoxylin and or DAPI Instrumentation Brightfield and or fluorescence microscope or scanner Safety Warnings and Precautions Formaldehyde is a poison and an irritant Avoid contact with skin and mucous membranes Use in a fume hood Ammonium hydroxide is highly volatile Use in a fume hood Xylene is both flammable and an irritant Avoid inhalation and contact with skin Use in a fume hood Probe Set Diluent QT PreAmplifier Mix QT and Amplifier Mix QT contain formamide a teratogen irritant and possible carcinogen Avoid contact with mucous membranes DAPI is a possible mutagen Avoid contact with skin and mucous membranes Perform all procedural steps in a well ventilated area at room temperature RT unless ot
14. plex assay can still be performed by assigning Fast Red to the housekeeping target and Fast Blue to the second target Brightfield detection of the Fast Blue signal for a medium expressing transcript could still be easily done while fluorescent detection would provide a more sensitive alternative for detecting a low expressing target tagged with Fast Blue Fluorescent Mode Guidelines The advantage of using alkaline phosphatase conjugated label probe for the enzymatic signal amplification is the availability of substrates with dual property such as Fast Red and Fast Blue which allows for both chromogenic and fluorescent detection of the targets However for a 2 plex assay in which both Label Probe 1 and Label Probe 6 are conjugated to the same alkaline phosphatase the enzymes conjugates are unable to differentiate between Fast Red and Fast Blue if both substrates are added simultaneously As a result the enzymatic signal amplification has to be performed sequentially in order to direct substrate color specificity to each target Additionally complete inactivation of the first alkaline phosphatase conjugated label probe LP6 AP is necessary especially when employing fluorescence mode for the detection of the targets Otherwise the residual LP6 AP activity can also convert Fast Red substrate in subsequent step into a red signal even at locations where TYPE 1 target is not present giving a false impression that the Fast Blue and Fast Red signals ar
15. D 2 Modified 1 Plex Protocol for Fast Blue Detection Using TYPE 1 or TYPE 6 Probe Sets Probe Set Modified Protocol Designation TYPE 1 A Perform the assay as directed through Step 15 Wash Slides on page 18 B Replace Label Probe 6 AP with Label Probe 1 AP in Step 16 Label Probe 6 AP Hybridization on page 19 C Continue with Step 17 Wash Slides to Step 19 Wash Slides on page 19 D Omit Step 20 Quench Label Probe 6 AP to Step 23 Apply Fast Red Substrate on page 20 E Continue with Step 24 Counterstain to Step 25 Mount and Image on page 21 TYPE 6 A Perform the assay as directed through Step 19 Wash Slides on page 19 B Omit Step 20 Quench Label Probe 6 AP to Step 23 Apply Fast Red Substrate on page 20 C Continue with Step 24 Counterstain to Step 25 Mount and Image on page 21 40 ViewRNA ISH Tissue 2 Plex Assay User Manual Using Frozen Tissues with ViewRNA ISH Tissue 2 Plex Assay About This Appendix Important Procedural Notes Modifications to Part 1 Sample Preparation and Target Probe Hybridization on page 42 About This Appendix This appendix provides procedural modification for running the ViewRNA ISH Tissue 2 Plex Assay on fresh frozen or OCT embedded frozen tissue sections This modified assay protocol has been tested on the following OCT embedded frozen tissue samples Bovine Ovary Human Colon skin testis Mouse Brain duodenum eye liver lung pancreas skin spinal cord
16. Dish green color 2 American Master Tech Scientific LWS20GR Tissue Tek Vertical 24 Slide Rack 1 Affymetrix QVC0503 American Master Tech Scientific LWSRA24 1000 mL glass beaker Major laboratory supplier Forceps Major laboratory supplier Pipettes P20 P200 P1000 Major laboratory supplier Hydrophobic Barrier Pen Affymetrix QVC0500 Vector Laboratories H4000 Mounting media a UltraMount Permanent Mounting Medium DAKO 1964 HistoMount Mounting Solution used only in Life Technologies 00 8030 conjunction with UltraMount a ADVANTAGE Mounting Medium Innovex NB300 Rectangular cover glass 24 mm x 55 mm VWR 48382 138 Affymetrix QVC0501 Aluminum foil Major laboratory supplier Double distilled water ddH 0 Major laboratory supplier 100 ethanol 200 proof VWR 89015 512 VWR 89125 188 Table 1 4 Required Materials Continued Chapter 1 Introduction Item Source Part Number 10X PBS pH 7 2 7 4 Calbiochem EMD or equivalent 6504 Gill s Hematoxylin I American Master Tech Scientific HXGHE1LT xylene or Histo Clear National Diagnostics or equivalent HS 200 37 formaldehyde EMD or equivalent FX0410 1 27 30 ammonium hydroxide VWR or equivalent JT9726 5 DAPI optional for fluorescence detection Life Technologies or equivalent D3571 Table 1 5 Required Equipment Item Source Part Number Either of the following hybridization systems ThermoBrite System 110 120 V and ThermoBrite Humidity Strips SUE
17. E 6 Target Table 4 12 Troubleshooting TYPE 1 Target Signals Observed in the Channel for TYPE 6 Target Probable Cause Recommended Action Spectral bleed through of Fast Red Check to make sure that the filter set for Fast Blue is as recommended signal Incorrect filter set for Fast Blue Use the correct filter set See Microscopy and Imaging Equipment Guidelines on page 6 signal for recommended filter set specifications for Fast Blue Co localized Fast Blue and Fast Red Signals When Using Only TYPE 6 Probe Set Table 4 13 Troubleshooting Co localized Fast Blue and Fast Red Signals When Using Only TYPE 6 in a 2 Plex Assay Probable Cause Recommended Action Residual LP6 AP activity Do not omit Step 20 on page 19 Quench Label Probe 6 AP Be sure to quench LP6 AP activity with AP Stop QT for the entire 30 min 30 ViewRNA ISH Tissue 2 Plex Assay User Manual Sample Pretreatment Optimization Procedures About Pretreatment Optimization Sample Pretreatment Optimization Setup Sample Preparation and Target Probe Hybridization on page 32 About Pretreatment Optimization Critical to any in situ assay is the balance between the adhesion of the tissue to the glass surface cross linking of the target molecules to the cellular structures by chemical fixatives and the subsequent unmasking of the RNA targets by heat treatment and protease digestion for the probes to hybridize For the ViewRNA ISH Tissue 2 Ple
18. Probe Set on page 29 Contacting Technical Support For technical support contact the appropriate resource provided below based on your geographical location Visit our website at www affymetrix com panomics for an updated list of FAQs and product support literature Table 4 1 Technical Support Contact Information Location Affymetrix North America Tel 1 877 726 6642 option 1 then option 3 E mail pqbhelp affymetrix com Europe Tel 43 1 7964040 120 E mail techQebioscience com Asia Tel 81 3 6430 430 E mail techsupport_asia affymetrix com 24 ViewRNA ISH Tissue 2 Plex Assay User Manual Weak or No Signals Table 4 2 Troubleshooting Weak or No Signal Probable Cause Recommended Action Incorrect pretreatment conditions Repeat pretreatment assay optimization procedure to determine optimal heat treatment time and protease digestion time that will strike a balance between morphology and signal Under pretreatment yields good morphology but poor signal due to insufficient unmasking of target Over pretreatment yields poor morphology and loss of signal due to over digestion Sample preparation Immediately place freshly dissected tissues in 2 20 volumes of fresh 1096 neutral buffered formalin NBF or 4 paraformaldehyde PFA at RT for 16 24 hours Tissue over fixed after protease digestion Make sure the tissue sections are not fixed for more than 5 min in 1096 NBF after proteas
19. The binding of these oligo pairs side by side to the target sequence serves as a base on which the signal amplification is built and is the core of the assay s sensitivity and specificity Using multiple pairs of oligos in a single probe set ensures that there are many opportunities for the probe to bind to the target s unmasked accessible regions so as to achieve the maximal signal amplification possible for that particular RNA target molecule When working with smaller targets or applications such as splice variants or RNA fusions the available number of oligo pairs in the probe set is naturally reduced and this will directly impact the sensitivity ofthe assay That is the probes will have fewer opportunities to find the unmasked areas of the target in order to generate signal at that location In these cases increasing the probe set concentration used in the assay from 1 40 to 1 30 or 1 20 might increase the sensitivity However note that there is always a general trade off between sensitivity and specificity Assigning Colors to Target mRNAs in 1 vs 2 Plex Assay The ViewRNA ISH Tissue 2 Plex Assay has multiplexing capability allowing in situ detection of up to two mRNA targets simultaneously using the ViewRNA TYPE 1 and or TYPE 6 probe sets The standard workflow of the assay is designed to automatically assign Fast Red signal to TYPE 1 and Fast Blue signal to TYPE 6 probe sets While both the Fast Red and Fast Blue signals that form are easil
20. VE CODIUOL ua dar Signe A BAR bane wel ai air eae ah t ER a gk 8 Heller don oa aan tia i a Aa ui e et ET 8 Sample Pretreatment Optimization nnana e 9 Assessment of Endogenous Alkaline Phosphatase 9 Probe Set Considerations 9 Assigning Colors to Target mRNAs in 1 vs 2 Plex Assay 10 Guidelines for Working with Tissue Microarrays 11 ViewRNA ISH Tissue 2 Plex Assay Procedure 13 About the ViewRNA ISH Tissue 2 Plex Assay Procedure 13 Important Procedural Notes and Guidelines 13 Essential Keys for a Successful Assay 13 Part 1 Sample Preparation and Target Probe Hybridization 14 Part 2 Signal Amplification and Detection 18 oi id dodo deco 23 0 6 0 Tro blesh otNg se Contacting Technical Support 2 23 anime dei he Soe uta Dc ac Da OY gatur 24 Weak or No Signals xcs ag 25 red Oe PEU a PER ER ata 26 Diff sed Signals 1 a ta 6 at Endogenous Alkaline Phosphatase Activity 26 Tissue Detachment From Slide
21. addition to the ViewRNA ISH Tissue 2 Plex Assay Kit ViewRNA TYPE 1 and TYPE 6 probe sets specific to your targets of interest must be purchased separately Probe sets are available in multiple sizes and should be stored at 20 C Refer to the package insert for quantities provided and design specificities Table 1 3 ViewRNA Probe Set and Storage Conditions Component Description Storage ViewRNA TYPE 1 Probe Set RNA specific oligonucleotides to your RNA target of 20 C interest TYPE 1 probe sets are compatible with the TYPE 1 Signal Amplification system which includes PreAmplifier Mix QT Amplifier Mix QT Label Probe 1 AP and Fast Red Substrate Refer to the package insert for design specificities ViewRNA TYPE 6 Probe Set RNA specific oligonucleotides to your target of interest 20 C TYPE 6 probe sets are compatible with the TYPE 6 Signal Amplification system which includes PreAmplifier Mix QT Amplifier Mix QT Label Probe 6 AP and Fast Blue Substrate Additional Required Materials and Equipment Table 1 4 and Table 1 5 list other materials and equipment that are required to perform the ViewRNA ISH Tissue 2 Plex Assay Do not substitute materials or suppliers Table 1 4 Required Materials Item Source Part Number Tissue Tek Staining Dish clear color 3 Affymetrix QVC0502 American Master Tech Scientific LWS20WH Tissue Tek Clearing Agent
22. ally means that in situ assays in general are only capable of relative and not absolute detection That is not every single molecule of a given target can be detected So in practice even if two RNA targets are theoretically expected to be co localized only a subset these two transcripts will be detected as being so due to lack of complete target accessibility Another factor that can limit the use of this assay for co localization studies is the nature of chromogenic assay and the sequential development of Fast Blue then Fast Red signals In chromogenic assay the enzyme converts the substrate into color precipitates and deposits them at the site where the RNA molecule is localized Because the Fast Blue and Fast Red substrates are sequentially developed in the ViewRNA ISH Tissue 2 Plex Assay the Fast Blue precipitates that are formed first and deposited have the potential to partially block subsequent hybridization of the TYPE 1 Label Probe by masking its binding sites on a nearby co localized target and consequently affecting the development of the Fast Red signal This is yet another form of accessibility issue that needs to be considered when performing co localization studies and analyzing the data obtained from such studies Consequently even when two targets are co localized only a subpopulation of the two is actually observed as such because of target accessibility be it at the probe hybridization step due to incomplete unmasking or at the
23. aration Guidelines on page 7 and optimized using the recommended pretreatment assay optimization procedure This table provides a reference or a starting point to minimize the number of test conditions if you do not have sufficient slides to perform the full recommended pretreatment optimization procedure Please note that the conditions listed here are specific to tissues prepared using 10 NBF and may not be applicable to tissue prepared using 496 PFA If you chose to use any of the pretreatment conditions listed in the lookup table it is important to include a negative control slide to assess whether the assay background is clean and cellular morphology is well defined Table B 1 Sample Pretreatment Optimization Lookup Table Tissue Information Optimal Conditions Min Range of Tolerance Min Species Type Heat treatment Protease at 40 C Heat treatment Protease at 90 95 C Human Brain 20 10 10 10 10 20 Breast 20 15 25 15 30 20 25 20 Colon 5 20 5 10 Kidney 20 10 Liver 20 20 10 20 Lung 10 20 Lymph node 10 20 Nasal polyp 5 5 Osteoarthritic 20 20 tissue Pancreas 10 10 10 20 5 10 Prostate 10 20 5 10 20 10 10 10 Salivary gland 10 10 5 10 Skin 5 10 Tonsil 10 20 Thyroid 10 20 Rat Kidney 10 20 10 10 20 20 Liver 10 20 Spleen 20 10 Thyroid 10 20 36 ViewRNA ISH Tissue 2 Plex Assay User Manual Table B 1 Sample Pretreatment O
24. ash the slides twice each time with 200 mL of 100 ethanol for 5 min with frequent agitation G Remove the slides from the rack and place them face up on a paper towel to air dry at RT for 5 min 4 Draw Hydrophobic A Dab the hydrophobic barrier pen on a paper towel several times before use to ensure proper Barrier flow of the hydrophobic solution 40 min B Tocreate a hydrophobic barrier 1 Place the slide over the template image making sure that the tissue sections fall inside the blue rectangle 2 Lightly trace the thick blue rectangle 2 4 times with the hydrophobic barrier pen to ensure a solid seal 3 Allow for barrier to dry at RT for 20 30 min Begin the next step while the barrier is drying 16 ViewRNA ISH Tissue 2 Plex Assay User Manual Table 3 1 ViewRNA ISH Tissue 2 Plex Assay Sample Preparation and Target Probe Hybridization Continued Step Action 5 HeatPretreatment A Tightly cover the beaker containing the 500 mL of 1X Pretreatment Solution with aluminum 10 25 min foil place it on a hot plate and heat the solution to a temperature of 90 95 C Use a Ed waterproof probe thermometer to measure and maintain the temperature of the solution at depending on 90 95 C during the pretreatment period Click g for a helpful video optimized time B Load the slides into the vertical slide rack C Using a pair of forceps submerge the slide rack into the heated 1X Pretreatment S
25. cedure to determine optimal heat treatment and protease digestion time Temperature of heat pretreatment condition too high Make sure the temperature is within the tolerance range of 90 95 C For fatty soft tissue such as breast adjust to 90 C high of a concentration Proteinase treatment is toolongorattoo Reduce proteinase concentration and or incubation time Poor Cell Morphology Table 4 7 Troubleshooting Poor Cell Morphology Probable Cause Recommended Action Incorrect pretreatment conditions Perform full pretreatment optimization procedure to determine optimal heat treatment and protease digestion time See Appendix A on page 31 Tissue sample not fixed properly Make sure that freshly dissected tissues are fixed in 10 NBF or 4 PFA for 16 24 hr Section thickness is variable or not optimal Make sure microtome is calibrated and tissue is sectioned at 5 1 um High Non Specific Binding on Glass Slide Table 4 8 Troubleshooting Non specific Binding on Glass Slide Probable Cause Recommended Action Incompatible glass slide a Use the recommended glass slides a Leica Non Clipped X tra Slide 1 mm White P N 3800200 or 3800210 o Fisherbrand Superfrost Plus Slides white label Fisher Scientific P N12 550 15 avoid other colored labels as they tend to give high background Prevalidate each new batch of slides by running the entire assay including probe
26. data gathered from the in situ assay can be analyzed to determine target expression Table C 1 Assessing Pretreatment Conditions Synpo and SPP1 Expression in Rat Kidney FFPE Tissue Heat Protease Brightfield Image Results Interpretation Pretreatment Digestion Time Min Time Min Untreated Morphology Reference Slide Probes Synpo and SPP1 Good morphology Intact cellular structure Good hematoxylin counterstaining of nuclei 0 0 Little or no signal dots observed Insufficient Pretreatment or Over Fixation of Tissue Probes Synpo and SPP1 Good morphology Intact cellular structure Strong hematoxylin counterstaining of nuclei 5 10 Weak diffused and non ubiquitous signal Few number of dots Optimal Pretreatment and Sample Preparations 4 Probes Synpo and SPP1 m Good morphology a Cellular structures and boundaries are retained and still identifiable 10 20 Good hematoxylin counterstaining of nuclei Strong punctated and ubiquitous signals in probe sample and clean background in probe sample 38 ViewRNA ISH Tissue 2 Plex Assay User Manual Table C 1 Assessing Pretreatment Conditions Synpo and SPP1 Expression in Rat Kidney FFPE Tissue Continued Heat Protease Brightfield Image Results Interpretation Pretreatment Digestion Time Min Time Min if 7 y Optimal Pretreatment and Sample Preparations vay i No Probes Fone h K _ Clean background j x a
27. dations for hybridization systems Ensure the hybridization system is appropriately humidified and that door lid is closed during hybridization steps Make sure the hybridization system is placed on a level bench Calibrate the hybridization system to 40 C using the ViewRNA Temperature Validation Kit Affymetrix QV0523 Prevent sections from drying out Prepare enough reagents and use the recommended volumes for each step of the assay Ensure that you have a solid seal when drawing your hydrophobic barriers Add all working reagents onto the slides before moving them to the 40 C hybridization system Tissue dries up during processing Keep tissue sections moist starting from the heat pretreatment step Add respective reagents immediately after decanting solution from the slides Keep tissue exposure to air as short as possible before adding hybridization reagents Add all working reagents onto the slides before moving them to the 40 C hybridization system Chapter 4 Troubleshooting 5 Table 4 2 Troubleshooting Weak or No Signal Continued Probable Cause Recommended Action Fast Red and Fast Blue Substrate solutions not freshly prepared Prepare Fast Red and Fast Blue Substrate solutions immediately before use Small targets splice variants or RNA fusions Doing one or both of the following may increase sensitivity but it should be noted that there is always a general tra
28. de off between specificity and sensitivity Increase probe set concentration by diluting target probe set 1 30 instead of 1 40 and hybridize for 2 hr Decrease hybridization temperature from 40 to 38 C Increase Fast Red incubation time to 45 min Probe set hybridization temperature time and or concentration not optimal Decrease hybridization temperature from 40 C to 38 C and increase the probe set concentration by diluting the target probe set 1 30 instead of 1 40 Hybridize for 2 hr Label Probe AP concentration too low Verify that the correct concentrations were used Increase the recommended concentration for Label Probe AP If this is necessary it may result in higher background Dark hematoxylin stain reduces visibility of the blue dots Tissues with lower cell density require longer hematoxylin incubation than tissues with higher cell density It may be helpful to titrate incubation times Increase the lamp brightness during viewing View under a 40X objective Image using fluorescent mode High Background Table 4 3 Troubleshooting High Background Probable Cause Recommended Action Tissue dries up during processing Prevent tissue sections from drying out after the pretreatment step Ensue that you have a solid seal when drawing your hydrophobic barrier Prepare enough reagents and use the recommended volume for each step of the assay Add respective rea
29. des to the hybridization system and incubate at 40 C for 15 min 22 Wash Slides A Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer 12 min B After incubation decant the working Label Probe 1 AP solution from the slides and insert them into the slide rack C Wash the slides 3 times each time with 200 mL of fresh Wash Buffer at RT for 3 min with constant and vigorous agitation 23 Apply Fast Red A Remove each slide and flick it to remove the Wash Buffer Without completely drying out the Substrate sections tap the slide on its edge and then wipe the backside on a laboratory wipe Place 45 m n slides face up on a flat elevated platform B Immediately add 400 uL of the AP Enhancer Solution to each tissue section and incubate at RT for 5 min while preparing the Fast Red Substrate C Prepare the Fast Red Substrate Add 5 ml of Naphthol Buffer and one Fast Red Tablet to a 15 ml conical tube Vortex at high speed to completely dissolve the tablet Protect from light until use by wrapping the tube in aluminum foil D Decant the AP Enhancer Solution and flick the slide twice to completely remove any excess AP Enhancer Solution Tap the slide on its edge then wipe the backside on a laboratory wipe Immediately add 400 uL of Fast Red Substrate onto each tissue section E Transfer the slides to the hybridization system and incubate at 40 C for 30 min F Insert an empty slide rack into a clear staining dish containi
30. e co localized For this reason it is absolutely necessary to quench any residual LP6 AP activity with the ViewRNA AP Stop QT prior to proceeding with the second label probe hybridization and development of the Fast Red color as this will ensure specific signals in fluorescent mode and brighter aqua blue dots in chromogenic mode Chapter 2 Assay Guidelines 1 Fast Red has a very broad emission spectrum and its bright signal that can bleed into adjacent Cy5 channel if one uses the standard Cy3 Cy5 filter sets for imaging For this reason it is critical that the recommended filter set for Fast Blue detection be used to avoid spectral bleed through of the Fast Red signal into the Fast Blue channel and interfering with Fast Blue detection Please refer to Table 1 7 on page 6 for exact filter set specifications Limitations of Chromogenic n Situ Assay in Co localization Studies When employing the ViewRNA ISH Tissue 2 Plex Assay for co localization studies it is crucial to understand the assay s strengths and limitations By definition a requisite for in situ detection is target accessibility While the assay with its branched DNA technology has the capability to detect RNA molecules down to single copy sensitivity and the probe sets are designed to maximize the binding opportunities to all accessible regions of the targets the overall detection for any given target is only as good as the unmasking of the target site is able to provide This essenti
31. e digestion RNA in tissue is degraded Verify tissue fixation Immediately place freshly dissected tissues in 2 20 volumes of fresh 1096 neutral buffered formalin NBF or 4 paraformaldehyde PFA for 16 24 hours at RT If fixation cannot be performed immediately be sure that the tissue is placed on dry ice or in liquid nitrogen to prevent RNA degradation Use positive control probe set s such as one for a housekeeping gene or a housekeeping gene panel ACTB GAPD and UBC to assess RNA integrity Reagents applied in wrong sequence Apply target probe sets PreAmplifier Mix QT Amplifier Mix QT Label Probe AP and substrates in the correct order Gene of interest not expressed Verify expression using other tissue lysate methods such as QuantiGene QuantiGene Plex assay or Affymetrix array Run the same probe set on known samples that have been validated to express the target of interest Incorrect storage condition Store the components at the storage condition as written on the component label or kit boxes Hybridization temperature not optimal Calibrate the hybridization system at 40 C using a ViewRNA Temperature Validation Kit Affymetrix P N QV0523 Mounting solution contained alcohol Use the recommended mounting media to mount your tissue see Step 25 Mount and Image on page 21 Avoid any mounting solution containing alcohol Tissue dries up during hybridization steps Recommen
32. e optimal pretreatment conditions in such case would be one that maximizes the number of cores with assay signal and minimizes the number of cores lost due to excessive heat treatment and protease digestion Due to their high cost and limited quantity TMAs would greatly benefit from the limited pretreatment optimization procedure since only as few as three slides might be necessary see Table B 2 on page 36 Assessment of Endogenous Alkaline Phosphatase The ViewRNA ISH Tissue 2 Plex Assay uses alkaline phosphatase to convert a chromogenic substrate into a colored signal For this reason it is important to assess the level of endogenous alkaline phosphatase AP activity in your tissue of interest prior to performing the assay Certain types of tissue such as stomach intestine placenta and mouse embryo are known to possess high levels of endogenous AP activity that can interfere with the assay While the problem is more prevalent in fresh frozen tissues it has also been observed in some FFPE samples To empirically determine the level of endogenous AP activity in your tissue type perform the pretreatment protocol as instructed for fresh frozen or FFPE tissue After the protease treatment and fixation in 10 NBF wash the samples in 1X TBS Sigma T5912 1L and incubate the sections with either Fast Blue Substrate or Fast Red Substrate If present endogenous AP can be inactivated with 0 2 M HC1 300 mM NaCl at RT for 15 min just before the pr
33. e section is covered with working protease solution It may be necessary to spread the solution with a pipette tip D Transfer the slides to the hybridization system and incubate at 40 C for the optimal time as determined in the Sample Pretreatment Optimization Procedures on page 31 E Pour 200 mL of 1X PBS into a clear staining dish and insert an empty slide rack into the dish F After the incubation decant the working protease solution from the slides insert the slides into the rack and wash gently but thoroughly by moving the rack up and down for 1 min G Repeat the wash one more time with another 200 mL of fresh 1X PBS H Transfer the slide rack to a clear staining dish containing 200 mL of 10 NBF and fix for 5 min at RT under a fume hood Wash the slides twice each time with 200 mL of fresh 1X PBS for 1 min with frequent agitation Chapter 3 ViewRNA ISH Tissue 2 Plex Assay Procedure 17 Table 3 1 ViewRNA ISH Tissue 2 Plex Assay Sample Preparation and Target Probe Hybridization Continued Step Action 7 Target Probe Set Hybridization 2 hr and 10 min A Prepare the working probe set solution using the table below as a guide Dilute the ViewRNA Probe Set 1 40 in prewarmed Probe Set Diluent QT and briefly vortex to mix Scale reagents according to the number of assays to be run and include one slide volume overage NOTE Add only 400 uL of Probe Set Diluent QT to the negative control or probe negative
34. f probe thermometer to measure and maintain the temperature of the solution at 90 95 C during the pretreatment period Click B for a helpful video B Setslide 1 aside on the lab bench C Load slides 9 and 10 into the vertical slide rack D Using a pair of forceps submerge the slide rack into the heated 1X Pretreatment Solution Cover the glass beaker with aluminum foil and incubate at 90 95 C for 10 min E Atthe end of the 10 min add slides 5 6 7 and 8 to the rack in the 90 95 C 1X Pretreatment Solution Cover the glass beaker with aluminum foil and incubate for 5 min F Atthe end of the 5 min add slides 2 3 4 into the rack in the 90 95 C 1X Pretreatment Solution Cover the glass beaker with aluminum foil and incubate for 5 min G After the pretreatment remove the slide rack with forceps submerge it into a clear staining dish containing 200 mL of ddH O and wash for 1 min with frequent agitation H Repeat the wash one more time with another 200 mL of fresh ddH O Transfer the slide rack to a clear staining dish containing 1X PBS IMPORTANT From this point forward do not let the tissue sections dry out Tissue sections that have been heat treated can be stored covered in 1X PBS at RT for up to one week Continue with Step 3 on page 33 Appendix A Sample Pretreatment Optimization Procedures 33 Table A 2 Sample Pretreatment Optimization Procedure Sample Preparation and Target Probe Set Hybridization Continued Step
35. fymetrix Inc assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information Copyright 2014 Affymetrix Inc All rights reserved Contents Chapter 1 Chapter 2 Chapter 3 Chapter 4 Contents i al a a d RE sa a d a a e un it 1 About This Manual cii eee cet ca ar et eee aaa a at qe cad 1 E a eLEPRRE MyPiE bebe dds 1 0 0 Assay OVeIVIeW Lope pee Du HEX CE ee DUC we a qe Wa ewe d a 2 Performance Highlights ecce Safety Warnings and Precautions 2 2 Required Materials and Equipment ViewRNA ISH Tissue 2 Plex Assay Kit 3 ViewRNA Probe Sets 4 Additional Required Materials and Equipment 4 Microscopy and Imaging Equipment Guidelines 6 Assay Guidelines 3s a uada Rope m Name PERS xa PR A CR e ed deaur 7 Tissue Preparation Guidelines 7 FFPE TMA Tissue Block Preparation 7 FFPE TMA Tissue Slide Preparation 7 Experiment Design Guidelines 0 0 0 2 es 8 ASSAY COMMONS 13e quede DEREN ol nan aad n AES Rene A 8 8 0 Negative Control POSITI
36. g Omit the target probe set A no probe negative control a Use a probe set designed to the sense strand of the target A more target specific negative control used to subtract assay background when assessing results Use a probe set for a target not present in your tissue sample A more general negative control used to subtract assay background when assessing results for example the bacterial gene dapB Positive Control Replicates This slide undergoes the entire assay procedure using a probe set against an ubiquitous or tissue specific target that has consistent medium high to high but not saturating expression level A positive control ensures that the assay procedure has been successfully run Examples of positive control targets include Housekeeping Genes ACTB GAPD or UBC Housekeeping Gene Panel A panel of several housekeeping genes can be pooled and used as a positive control whenever the expression level of any one given housekeeping gene is unknown in the tissue of interest For example pool ACTB GAPD and PPIB probe sets at equal volumes to form a panel and then dilute the panel of probe sets 1 40 to create a working probe set solution for use as a positive control We recommend running all assays in duplicate Chapter 2 Assay Guidelines 9 Sample Pretreatment Optimization The pretreatment of tissue sections is critical for the success of all in situ assays Pretreatment for the ViewRNA T
37. ge 18 About the ViewRNA ISH Tissue 2 Plex Assay Procedure ViewRNA ISH Tissue 2 Plex Assay can be run in a single long day or broken up over two days for added flexibility The procedure includes two parts Part 1 Sample Preparation and Target Probe Set Hybridization optional stopping point Part 2 Signal Amplification and Detection Important Procedural Notes and Guidelines The procedure assumes running a maximum of 12 slides at a time and that the size of the section does not exceed the maximum coverage area recommended Do not mix and match kit components from different lots Before beginning the procedure know the optimized conditions heat treatment time and protease digestion time for your sample type If you do not know these optimized conditions refer to Appendix A Sample Pretreatment Optimization Procedures on page 31 Throughout the procedure dedicate the Tissue Tek staining dishes as follows Clear staining dish for Formaldehyde u Green staining dish for Gill s hematoxylin Green staining dish for xylene Histo Clear The remaining two clear staining dishes can be used interchangeably for 1X PBS 100 ethanol Wash Buffer ddH O Storage Buffer and DAPI Rinse staining dishes between steps with ddH O If using a humidified tissue culture incubator without CO as the hybridization system o Verify that the water jacket or bottom tray is filled with water o Use an aluminum slide rack to t
38. gents immediately after decanting solution from the slides Keep tissue exposure to air as short as possible before adding hybridization reagents Make sure that the hybridization system is appropriately humidified Make sure the hybridization system is set at 40 C and that the lid door is closed during hybridization steps Process as few or as many slides at a time as you are comfortable doing Incomplete removal of paraffin Use fresh xylene or Histo Clear solution Immediately submerge the warm slides into the Histo Clear solution after baking Insufficient washing Move the slide rack up and down with constant and vigorous agitation Click gD for a helpful video a Increase wash incubation time by 1 min per wash Hybridization temperature not optimal Calibrate the hybridization system at 40 C using the ViewRNA Temperature Validation Kit Affymetrix P N QV0523 Concentration of hybridization reagents too high Double check the dilution calculation for all working solutions Suboptimal pretreatment conditions Perform the pretreatment optimization procedure to determine the optimal heat treatment and protease digestion time Label Probe AP concentration too high a Verify that the correct concentrations were used Decrease the recommended concentration for Label Probe AP 26 ViewRNA ISH Tissue 2 Plex Assay User Manual Diffused Signals Table 4 4 Troubleshooting Diffused Signal
39. herwise noted Discard all reagents in accordance with local state and federal laws Required Materials and Equipment The ViewRNA ISH Tissue 2 Plex Assay uses the following items each sold separately and available in multiple sizes ViewRNA ISH Tissue 2 Plex Assay Kit see Table 1 2 QVT0012 24 assays QVT0013 96 assays ViewRNA TYPE 1 and TYPE 6 probe sets see Table 1 3 Table 1 4 and Table 1 5 list additional material and equipment requirements for the assay Chapter 1 Introduction 3 ViewRNA ISH Tissue 2 Plex Assay Kit The kits are configured for processing a minimum of 6 assays 24 assay kit or 12 assays 96 assay kit respectively per experiment Table 1 2 lists the components of the ViewRNA ISH Tissue 2 Plex Assay Kit and their recommended storage conditions Refer to the component package insert for the quantity of individual components supplied Kits are shipped in two parts based on storage conditions and have a shelf life of six months from the date of delivery when stored as recommended Table 1 2 ViewRNA ISH Tissue 2 Plex Assay Kit Components and Storage Conditions Wash Comp 2 aIMPORTANT Do not freeze Component Description Storage 100X Pretreatment Solution Aqueous buffered solution 2 8 C Protease QF Enzyme in aqueous buffered solution 2 8 C Probe Set Diluent QT Aqueous solution containing formamide detergent and bloc
40. iner G Prepare 1 L of 0 01 ammonium hydroxide Work in a fume hood Add 0 33 mL 30 ammonium hydroxide and 999 67 mL ddH 0 in a 1 L capacity container H Ensure the availability of 600 mL 100 ethanol 1 4 L ddH O 600 mL xylene or 400 mL Histo Clear a 200 mL Gill s Hematoxylin 200 mL of 3 ug mL DAPI in 1X PBS optional for fluorescence detection Store in the dark at 4 C until use Thaw probe set s Mix briefly centrifuge to collect contents and place on ice until use J Prewarm 40 mL 1X PBS and Probe Set Diluent QT to 40 1 C K Optional If performing both parts of the assay in 1 day Prewarm PreAmplifier Mix QT Amplifier Mix QT and Label Probe Diluent QF to 40 C Briefly spin down the Label Probe 1 AP Label probe 6 AP and Blue reagents then place on ice until use Bring Fast Red Tablets Napthol Buffer Blue Buffer and AP Enhancer Solution to RT Chapter 3 ViewRNA ISH Tissue 2 Plex Assay Procedure 15 Table 3 1 ViewRNA ISH Tissue 2 Plex Assay Sample Preparation and Target Probe Hybridization Continued Step Action 2 Prepare Buffers L Optional If using a microplate shaker for the washes optional follow the steps below Reagents and Click Bm for a helpful video Equipment While 1 Setthe speed to 550 rpm Slides Bake 2 Place a slide rack in a clear staining dish containing the appropriate reagent and insert continued the slides into the rack 3 Manually lif
41. issue 2 Plex Assay consists of heat treatment and protease digestion These pretreatment steps help to unmask the RNA targets allowing for better probe accessibility and thereby increasing assay signal However excessive pretreatment can have a negative effect on tissue morphology Thus we recommend optimizing the pretreatment conditions for each new tissue type see Appendix A Sample Pretreatment Optimization Procedures on page 31 Once the optimal pretreatment conditions are determined they can generally be used for most targets within the particular tissue In instances when the transcript is particularly rare or expressed at an extremely low level the optimal pretreatment condition may need to be one that favors signal over morphology Refer to the Sample Pretreatment Lookup Table on page 35 for heat treatment and protease conditions that we have found to be optimal for a number of tissues prepared according to the recommended guidelines in this manual using 10 NBF This table serves as a reference or starting point only and may not be applicable to tissues prepared using 4 PFA If you do not obtain the desired results we recommend performing either the full or limited Sample Pretreatment Optimization Procedures on page 31 depending on availability of your samples When optimizing pretreatment conditions for TMAs it is important to understand that it is impossible to identify one condition that is ideal for every tissue type on the array Th
42. k Cy7 B Alexa 750 filter modified with excitation filter FF02 628 40 25 Compatible to above Assay Guidelines Tissue Preparation Guidelines Experiment Design Guidelines on page 8 Sample Pretreatment Optimization on page 9 Assessment of Endogenous Alkaline Phosphatase on page 9 Probe Set Considerations on page 9 Assigning Colors to Target mRNAs in 1 vs 2 Plex Assay on page 10 Guidelines for Working with Tissue Microarrays on page 11 Tissue Preparation Guidelines This section provides critical guidelines for preparation of FFPE tissue blocks FFPE tissue slides and tissue microarray TMA slides for use with the ViewRNA ISH Tissue 2 Plex Assay Samples prepared outside of these guidelines may not produce the best results FFPE TMA Tissue Block Preparation Immediately place freshly dissected tissues in 2 20 volumes of fresh 1096 neutral buffered formalin NBF or 496 paraformaldehyde PFA at room temperature RT for 16 24 hr Trim larger specimens to 3 mm thickness to ensure faster diffusion of the fixative into the tissue Rinse dehydrate and embed in a paraffin block Store FFPE tissue blocks at RT FFPE TMA Tissue Slide Preparation Section FFPE tissue to a thickness of 5 1 um If working with TMAs core size should be gt 1 0 mm diameter Maximum tissue area is 20 mm x 30 mm and should fit within the hydrophobic barrier Mount sections as shown in Figure 2 1 on page 8 onto one ofthe fol
43. ker 2 8 C Label Probe Diluent QF Aqueous solution containing detergent 2 8 C PreAmplifier Mix QT DNA in aqueous solution containing formamide and detergent 2 8 C Amplifier Mix QT DNA in aqueous solution containing formamide and detergent 2 8 C Label Probe 6 AP Alkaline phosphatase conjugated oligonucleotide in aqueous buffered solution 2 8 C Blue Buffer Buffer required for preparation for Blue Substrate 2 8 C Blue Reagent 1 Blue precipitating substrate component 1 for the detection of alkaline phosphatase 2 8 C activity Blue Reagent 2 Blue precipitating substrate component 2 for the detection of alkaline phosphatase 2 8 C activity Blue Reagent 3 Blue precipitating substrate component 3 for the detection of alkaline phosphatase 2 8 C activity AP Enhancer Solution Aqueous buffered solution 2 8 C Fast Red Tablets Red precipitating substrate for the detection of alkaline phosphatase activity 2 8 C Naphthol Buffer Buffer required for preparation of Red Substrate 2 8 C Label Probe 1 AP Alkaline phosphatase conjugated oligonucleotide in aqueous buffered saline 2 8 C AP Stop QT Aqueous buffered solution intended for the inactivation of residual LP6 AP activity 15 30 C after the Fast Blue substrate development Wash Buffer Component 1 Aqueous solution containing detergent 15 30 C Wash Comp 1 Wash Buffer Component 2 Aqueous buffered solution 15 30 C 4 ViewRNA ISH Tissue 2 Plex Assay User Manual ViewRNA Probe Sets In
44. label probe hybridization step due to masking of the binding site by the deposition of the Fast Blue precipitates Guidelines for Working with Tissue Microarrays Process TMA slides using the same assay procedures but with the following two modifications Increase the initial baking step time from 60 to 90 min This additional baking time will increase the tissue attachment to the slide reducing the risk of small gt 1 mm core sections falling off during assay procedure a Increase the volume slide of the protease working solution to prevent tissues at the edge of the TMA from drying out When designing TMAs to be used in the ViewRNA ISH Tissue 2 Plex Assay it is important to understand that only one optimized condition can be used when running the assay Therefore if you want multiple tissue types within the same TMA block we recommend running an optimization procedure on each individual FFPE tissue type to identify the most favorable pretreatment boiling and protease condition Based on the optimal condition of the tissue morphology signal strength and residual cores you can judge if there is one optimization condition that will be suitable for all of the sample types 12 ViewRNA ISH Tissue 2 Plex Assay User Manual ViewRNA ISH Tissue 2 Plex Assay Procedure About the ViewRNA ISH Tissue 2 Plex Assay Procedure Part 1 Sample Preparation and Target Probe Hybridization on page 14 Part 2 Signal Amplification and Detection on pa
45. ling film to prevent evaporation B Discard 1X Pretreatment Solution 1096 NBF remaining protease and probe set working solutions C Store the remaining 1X PBS and Wash Buffer at RT for use in Part 2 Signal Amplification and Detection on page 18 If using a ThermoBrite System rewet the ThermoBrite Humidity Strips in ddH O E Proceed to Step 10 Prepare Additional Buffers and Reagents on page 18 when you are ready to continue the assay 18 ViewRNA ISH Tissue 2 Plex Assay User Manual Part 2 Signal Amplification and Detection Table 3 2 ViewRNA ISH Tissue 2 Plex Assay Signal Amplification and Detection Step Action 10 Prepare Additional A Pour Gill s Hematoxylin into a clear staining dish and store at RT protected from light until Buffers and use Reagents B Prewarm PreAmplifier Mix QT Amplifier Mix QT and Label Probe Diluent QF buffers to 40 C 5 min C Briefly spin down the Label Probe 1 AP Label probe 6 AP and Blue reagents Place them on ice D Bring Fast Red Tablets Naphthol Buffer AP Enhancer Solution and Blue Buffer to RT 11 Wash Slides A Remove the slides from Storage Buffer 5 min B Wash the slides 2 times each time with 200 mL of fresh Wash Buffer at RT for 2 min with constant and vigorous agitation 12 PreAmplifier A Swirl the PreAmplifier Mix QT bottle briefly to mix the solution Hybridization B Remove each slide and flick it to remove the Wash Buffer Without com
46. lowing types of positively charged glass slides a Leica Non Clipped X tra Slides 1 mm white P N 3800200 in U S Canada and Asia Pacific regions or P N 3800210 in Europe Fisherbrand Superfrost Plus Slides white label Fisher Scientific P N 12 550 15 Avoid other colored labels as they tend to give high background a Air dry freshly mounted sections at RT overnight or at 37 C for 5 hr Bake slides at 60 C for 1 hr to immobilize tissue sections Storage n Short term Store sections in a slide box at RT for up to 2 weeks o Long term Store sections in a slide box at 20 C for up to 1 year avoid freeze thaw a Slides can be shipped at the temperature at which they were originally stored NOTE See Guidelines for Working with Tissue Microarrays on page 11 for more information 8 ViewRNA MISH Tissue 2 Plex Assay User Manual Figure 2 1 Correct Tissue Section Placement on Glass Side 3mm 12 mm 7 mm j3mm Place tissue sections in this area Experiment Design Guidelines Assay Controls We recommend running one positive and one negative control slide based on your sample type in every ViewRNA ISH Tissue 2 Plex Assay This will allow you to qualify and interpret your results Negative Control This slide undergoes the entire assay procedure and assesses the assay background from different levels The negative control can be one of the followin
47. ng 200 mL of 1X PBS G After incubation decant the Fast Red Substrate solution from the slides and insert them into the slide rack H Rinse off the excess Fast Red Substrate from the slides by moving the slide rack up and down for 1 min Chapter 3 ViewRNA ISH Tissue 2 Plex Assay Procedure 21 Table 3 2 ViewRNA ISH Tissue 2 Plex Assay Signal Amplification and Detection Continued Step Action 24 Counterstain A Transfer the slide rack to the clear staining dish containing the 200 mL of Gill s hematoxylin 25 min and stain for 5 10 sec at RT B Wash the slides 3 times each time with 200 mL of fresh ddH O for 1 min by moving the slide rack up and down C Pour off the ddH O refill with 200 mL of 0 01 ammonium hydroxide and incubate the slides for 10 seconds Unused 0 01 ammonium hydroxide can be stored at RT for up to 1 month D Wash the slides once more in 200 mL of fresh ddH O by moving the rack up and down for 1 min E Optional If you plan to view slides using a fluorescent microscope move the slide rack into a clear staining dish containing 200 mL DAPI 3 ug mL Stain the slides for 1 min then rinse them in 200 mL of fresh ddH O by moving the slide rack up and down for 1 min F Remove the slides from the slide rack and flick to remove the excess ddH O Tap the slide on its edge then wipe the backside on a laboratory wipe Place them face up onto a paper towel to air dry in the dark G Ensure that slide section
48. obe hybridization but after the sample has undergone protease treatment 10 NBF fixation and 2 washes in 1X PBS Probe Set Considerations Probe sets of the same TYPE can be combined to create a target panel pan or cocktail For example identifying epithelial cells could be easily accomplished by pooling different cytokeratin probe sets of the same type such as TYPE 1 KRT5 KRT7 KRT8 KRT10 KRT19 KRT19 and KRT20 into a single assay However we do not recommend combining more than 10 targets for any one signal amplification system be it TYPE 1 or TYPE 6 How the probe sets are diluted to generate a panel depends on the application For example if the goal is to identify all of the epithelial cells or to assess RNA integrity then each probe set can be diluted 1 40 However when using a panel of housekeeping gene probe sets for optimizing pretreatment conditions the probe sets e g ACTB GAPD and PPIB should be pooled at equal volumes to form the panel and then diluted 1 40 to create the working probe set solution This ensures that the panel expression is sufficiently high but not saturated so that the differences in signal between pretreatment conditions can be distinguished 10 ViewRNA ISH Tissue 2 Plex Assay User Manual The typical design for a ViewRNA Probe Set consists of 40 unlabeled oligos or 20 pairs of oligos per RNA target and spans approximately 1000 bases of the target transcript to achieve maximal sensitivity
49. olution Cover the glass beaker with aluminum foil and incubate at 90 95 C for the optimal time as determined in Sample Pretreatment Optimization Procedures on page 31 D After pretreatment remove the slide rack with forceps submerge it into a clear staining dish containing 200 mL of ddH O and wash for 1 min with frequent agitation Repeat the wash one more time with 200 mL of fresh ddH O F Transfer the slide rack to a clear staining dish containing 1X PBS IMPORTANT Do not let the tissue sections dry out from this point forward After heat pretreatment sections can be stored covered in 1X PBS at RT overnight 6 Protease Digestion A Prepare the working protease solution using the table below as a guide Dilute the Protease and Fixation QF 1 100 in prewarmed 1X PBS and briefly vortex to mix Scale reagents according to the number of assays to be run Include one slide volume overage 30 50 min depending on optimized time Working Protease Solution per Slide Reagent Volume Protease QF 4 uL 1X PBS prewarmed to 40 C 396 uL Total volume 400 uL B Remove each slide and flick it to remove excess 1X PBS Without completely drying out the sections tap the slides on the edge and then wipe the backside on a laboratory wipe C Place the slides face up on a flat elevated platform e g Eppendorf tube rack for easier handling and immediately add 400 uL of the working protease solution onto the tissue section Make sure that the tissu
50. on ice Bring Fast Red Tablets Napthol Buffer Blue Buffer and AP Enhancer Solution to RT Prepare 1 Lof 0 01 ammonium hydroxide Work in a fume hood Add 0 33 mL 30 ammonium hydroxide and 999 67 mL ddH20 in a 1 L capacity container K Optional If using a microplate shaker for the washes set the speed to 550 rpm Click for a video with detailed instructions 4 Wash Slides Remove slide rack from the 1096 NBF and wash the slides twice each time with 200 mL of 1X PBS for 1 min with frequent agitation 5 Tissue Dehydration A Dehydrate the tissue by sequentially soaking the rack of slides in 50 70 and then 100 ethanol in a clear staining dish each time at RT for 10 min without agitation B Remove the slide rack from the 10096 ethanol and drain the excess on a paper towel C Transfer the entire rack of slides to a 60 C dry incubator oven and bake the slides for 60 min Note Following the baking step continue with the assay within 1 hr beginning with Step 4 Draw Hydrophobic Barrier on page 15 and skipping to Step 6 Protease Digestion and Fixation on page 16 The heat treatment step is NOT REQUIRED for frozen tissues
51. ons for some common tissues If samples are limited see Table B 2 on page 36 Table A 1 Pretreatment Optimization Setup Protease Incubation Heat Pretreatment Time min Time min 0 5 10 20 0 Slide 1 Morphology reference 10 Slide 2 Slide 5 Slide 9 20 Slide 3 Slide 6 Slide 10 Slide 7 No Probe Control 40 Slide 4 Slide 8 32 ViewRNA M ISH Tissue 2 Plex Assay User Manual Before starting the pretreatment optimization protocol please read the sections on mportant Procedural Notes and Guidelines on page 13 and Essential Keys for a Successful Assay on page 13 The pretreatment optimization procedure for the ViewRNA ISH 2 Plex Tissue is divided into two parts that can be performed in a single day or over two days Part 1 Sample Preparation and Target Probe Set Hybridization optional stopping point Part 2 Signal Amplification and Detection We do not recommend stopping the procedure at any point in the assay unless specifically indicated Sample Preparation and Target Probe Hybridization Table A 2 Sample Pretreatment Optimization Procedure Sample Preparation and Target Probe Set Hybridization Step Action 1 Bake Slides See Step 1 to Step 4 starting on page 14 2 HeatPretreatment A Tightly cover the beaker containing the 500 mL of 1X Pretreatment Solution with aluminum 10 25 min foil place it on a hot plate and heat the solution to a temperature of 90 95 C Use a waterproo
52. pletely drying out the 35 min sections tap the slide on its edge and then wipe the backside on a laboratory wipe Place the slides face up on a flat elevated platform and immediately add 400 uL of PreAmplifier Mix QT to each tissue section C Transfer slides to the hybridization system and incubate at 40 C for 25 min 13 Wash Slides A Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer 8 min B After incubation decant the PreAmplifier Mix QT from the slides and insert them into the slide rack C Wash the slides 3 times each time with 200 mL of fresh Wash Buffer at RT for 2 min with constant and vigorous agitation 14 Amplifier A Swirl the Amplifier Mix QT bottle briefly to mix the solution Hybridization B Remove each slide and flick it to remove the Wash Buffer Without completely drying out the 20 min sections tap the slide on its edge and then wipe the backside on a laboratory wipe Place the slides face up on a flat elevated platform and immediately add 400 uL of Amplifier Mix QT to each tissue section C Transfer slides to the hybridization system and incubate at 40 C for 15 min 15 Wash Slides A Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer 8 min B After incubation decant the Amplifier Mix QT from the slides and insert them into the slide rack C Wash the slides 3 times each time with 200 mL of fresh Wash Buffer at RT for 2 min with constant and vigorou
53. ptimization Lookup Table Continued Tissue Information Optimal Conditions Min Range of Tolerance Min Species Type Heat treatment Protease at 40 C Heat treatment Protease at 90 95 C Mouse Bone 20 20 Brain 10 10 Heart 10 40 20 20 Kidney 20 20 10 20 Liver 20 20 5 40 10 20 Lung 10 20 Retina 10 10 Salmon Heart 10 10 Muscle 10 20 Monkey Mucosal rectum 10 20 If your tissue type is not listed in Table B 1 and you have only limited slides available for the pretreatment optimization Table B 2 provides the recommended heat treatment and protease incubation times that will likely give the best chance of achieving an acceptable pretreatment conditions for your ViewRNA ISH Tissue 2 Plex Assay Table B 2 Heat Treatment and Protease Incubation Times for Limited Optimization Number of Heat Treatment Time min Protease Time min Available Slides 3 5 10 10 10 10 20 5 5 10 5 20 10 10 10 20 20 10 7 5 10 5 20 10 10 10 2 20 10 20 20 0 0 Evaluating Results Assessing Pretreatment Conditions Analyzing Target Expression on page 38 Assessing Pretreatment Conditions This section provides sample images obtained from the ViewRNA ISH Tissue 2 Plex Assay performed on rat kidney tissue to illustrate the effects of optimal and suboptimal pretreatment conditions on Arbp signal strength versus morphology and to demonstrate how
54. ransfer slides to the incubator Do not leave the incubator door open longer than necessary when transferring slides particularly during the protease optimization procedure This will help maintain the required temperature Typical processing times included in the assay procedure assume that the preparations for the following step are being done during the incubation periods Essential Keys for a Successful Assay Prepare samples following Tissue Preparation Guidelines on page 7 Organize the preparation of the assay before you start o Verify that all materials and equipment are available u Be mindful of the incubation times temperatures as variations can negatively affect assay signal and background o Double check all reagent calculations as correct reagent volumes and concentrations are critical Employ good washing techniques Frequently washing is performed too gently Adequate washing is important for consistent low backgrounds Click for a helpful video Calibrate temperatures for hybridization system to 40 C and dry oven to 60 C using the ViewRNA Temperature Validation Kit Ensure that hybridization system is appropriately humidified 14 ViewRNA ISH Tissue 2 Plex Assay User Manual a DO NOT let tissues dry out where indicated in the procedure Click g for a helpful video Incorporate controls both positive and negative so that results are unambiguous and can be interpreted See Experiment Design G
55. s Probable Cause Recommended Action Tissue dries up during processing Prevent tissue sections from drying out after the pretreatment step Ensure that you have a solid seal when drawing your hydrophobic barrier Prepare enough reagents and use the recommended volume for each step of the assay Add respective reagents immediately after decanting solution from the slides Limit tissue exposure to air before adding hybridization reagents Make sure that the hybridization system is appropriately humidified Make sure the hybridization system is set at 40 C and that the lid door is closed during hybridization steps Process as few or as many slides at a time as you are comfortable doing Incomplete removal of AP Enhancer Ensure that excess AP Enhancer is removed by decanting the AP Enhancer and flicking the slides twice prior to adding Fast Red Substrate Insufficient washing Make sure tissues are washed twice in 1X PBS after protease digestion and twice again after subsequent fixing in 1096 NBF Fast Red Substrate and Fast Blue Substrate solutions not freshly prepared Prepare Fast Red and Fast Blue Substrate solutions immediately before use Slides are not dried before mounting Ensure that the sections are completely dry 20 min before mounting Mounting solution contained alcohol Use the recommended mounting media to mount your tissue see Step 25 Mount and Image on page 21
56. s agitation Chapter 3 ViewRNA ISH Tissue 2 Plex Assay Procedure 19 Table 3 2 ViewRNA ISH Tissue 2 Plex Assay Signal Amplification and Detection Continued Step Action 16 Label Probe 6 AP A Briefly vortex and spin down Label Probe 6 AP before using Hybridization B Prepare the working Label Probe 6 AP solution using the table below as a guide Dilute Label 20 min Probe 6 AP 1 1000 in prewarmed Label Probe Diluent QF and briefly vortex to mix Scale reagents according to the number of assays to be run and include one slide volume overage Working Label Probe 6 AP Solution per Slide Reagent Volume Label Probe Diluent QF prewarmed to 40 C 399 6 uL Label Probe 6AP 0 4 uL Total volume 400 uL C Remove each slide and flick to remove the Wash Buffer Without completely drying out the sections tap the slide on its edge and then wipe the backside on a laboratory wipe Place the slides face up on a flat elevated platform and immediately add 400 uL of working Label Probe 6 AP solution to each tissue section D Transfer the slides to the hybridization system and incubate at 40 C for 15 min 17 Wash Slides A Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer 12 min B After incubation decant the working Label Probe 6 AP solution from the slides and insert them into the slide rack C Wash the slides 3 times each time with 200 mL of fresh Wash Buffer at RT
57. s are completely dry before mounting 20 min 25 Mount and Image 40 min If using DAKO Ultramount mounting medium For no coverslipping 20X viewing or imaging A B C E Place the slide flat on a counter top with specimen facing up Dab the first 2 3 drops of Ultramount onto a paper towel to remove bubbles Apply a sufficient amount of Ultramount to completely cover the specimen with a thin layer 3 4 drops of mounting medium Place slides horizontally in a 70 C oven incubator to dry the mounting medium Allow 10 30 min for the mounting medium to harden completely The drying time depends on the amount of mounting medium applied Image or store slides at RT For post mounting with coverslip crisper 20X or 40X viewing or imaging lt ON Work in a fume hood and follow the no coverslipping procedure Make sure that the Ultramount is completely hardened Allow the slides to come to RT Apply HistoMount directly on top of the dried Ultramount Place coverslip on top and allow to air dry at RT for 15 min Image or store slides at RT If using ADVANTAGE mounting medium A mons Place a 24 mm x 55 mm cover glass horizontally onto a clean flat surface Dab the first 2 3 drops of mounting media onto a paper towel to remove bubbles Add 2 drops of the ADVANTAGE medium directly onto the middle of the cover glass Use a pipette tip to draw out any air bubbles in the droplets Inver
58. s id e Acceptable morphology and cellular architecture K if t Good hematoxylin counterstaining of nuclei Sa a 7 10 20 r We E s Y f r Li 4 x LA re Se F 2 ni gt gt x V Over Pretreatment or Under Fixation Probes Synpo and SPP1 Poor morphology Loss of cellular structure and boundaries due to excessive heat treatment and protease digestion 10 40 Poor hematoxylin counterstaining of nuclei Weak signal and fewer number of dots Analyzing Target Expression Each observable punctated dot represents a single RNA molecule within the cell that the ViewRNA ISH Tissue 2 Plex Assay is able to detect assuming the RNA target is intact and properly unmasked for the probe to access These dots are typically uniform in size However smaller than average size dots can also be present and this usually indicates that the transcript is not properly unmasked or that the RNA target is not intact resulting in the binding of only one or a few pairs of oligonucleotides from the probe set Conversely a larger than average size dot can occur when multiple targets are found clustered in the same physical area Naturally with everything being equal an RNA target with a low expression will yield fewer numbers of dots than one with a high expression In quantifying the results to assess the RNA target expression it is important to consider the pattern and number of dots observed in the Negative Control such
59. set on empty slides with hydrophobic barriers without fixed tissues to determine if the slides are suitable for the assay Insufficient washing Move the slide rack up and down with constant and vigorous agitation Click for a helpful video Increa ash incubation time by 1 min per wash Concentration of hybridization reagents was too high Confirm that the dilution calculations are correct for all working solutions 28 ViewRNA ISH Tissue 2 Plex Assay User Manual Pink Non Specific Background Where Paraffin Was Table 4 9 Troubleshooting Pink Non Specific Background Where Paraffin Was Probable Cause Recommended Action Incomplete removal of paraffin Be sure to use fresh Histo Clear or xylene for the indicated amount of time during the dewaxing step a Use 3 changes of Histo Clear instead of 2 changes Polymerization of poor quality paraffin Melt the paraffin at 80 C for 3 min and remove paraffin using 3 changes of fresh Histo Clear Do not bake the slides at a temperature higher than 60 C Hydrophobic Barrier Falls Off Table 4 10 Troubleshooting the Hydrophobic Barrier Probable Cause Recommended Action Incompatible glass slide a Use the recommended glass slides a Leica Non Clipped X tra Slide 1 mm White P N 3800200 or 3800210 a Fisherbrand Superfrost Plus Slides white label Fisher Scientific P N12 550 15 avoid other colored labels as they tend
60. sh and insert an empty slide rack into the solution Insert Overnight frozen tissue slides into the slide rack and incubate at 4 C for 16 18 hr 3 Prepare Buffers A Verify that the hybridization system is set to 40 1 C and appropriately humidified Reagents and B Prepare 2 L 1X PBS Add 200 mL 10X PBS and 1 8 L ddH 0 to a 2 L capacity container Equipment C Prepare 200 mL of 50 ethanol Add 100 mL 100 ethanol and 100 mL ddH O to a 200 mL capacity container D Prepare 200 mL of 7096 ethanol Add 60 mL 10096 ethanol and 140 mL ddH20 to a 200 mL capacity container E Prepare 4 L Wash Buffer Add the components below in the order listed to a 4 L capacity container and mix well 3L ddH O 36 mL Wash Comp 1 10 mL Wash Comp 2 Adjust the total volume to 4 L with ddH O F Prepare 200 mL Storage Buffer for optional stopping point Add 60 mL Wash Comp 2 and 140 mL ddH 0 to a 200 mL capacity container G Ensure availability of 1000 mL ddH O 200 mL Gill s Hematoxylin 200 mL of 3 ug mL DAPI in 1X PBS optional for fluorescent detection store in the dark at 4 C until use H Thaw probe set s Mix briefly centrifuge to collect content and place on ice until use l Prewarm 10 mL of 1X PBS and Probe Set Diluent QF to 40 C J Optional for 1 day assay Prewarm PreAmplifier Mix QT Amplifier Mix QT and Label Probe Diluent QF to 40 C Briefly spin down the Label Probe 1 AP Label Probe 6 AP and Blue Reagents Place
61. t Blue Aqua blue Far red Nucleus Hematoxylin DAPI Light purple blue Blue Viewing and Digital Capturing Options Microscope Type Recommended Microscope System Required Optics Recommended Filters Brightfield viewing Standard brightfield microscope Leica DM series Nikon E series Olympus BX series Zeiss Axio Lab Scope Imager a Or equivalent Requires 20 and 40X objectives Requires neutral density filters and or color filters for white balancing Fluorescence viewing and image capture Microscope with camera and fluorescence options Verify that the camera does not have infrared blocking filter Leica DMA series Nikon E series Olympus BX series Zeiss Axio Lab Scope Imager Or equivalent Requires 20 and 40X objectives Numerical aperture NA gt 0 5 For Fast Red Substrate use Cy3 TRITC filter set Excitation 530 20 nm Emission 590 20 nm Dichroic 562 nm For Fast Blue Substrate use custom filter seta Excitation 630 20 nm Emission 775 25 nm Dichroic 750 nm For DAPI filter set Excitation 387 11 nm Emission 447 60 nm Automated image capture in brightfield and or fluorescence mode Digital pathology scanner system Aperio ScanScope AT XT CS use FL version for fluorescence Leica SCN400 F Olympus Nanozoomer RS Or equivalent Recommend scanning at 40X when expression is low aRecommended vendor Semroc
62. t the rack up and down 10 times Put the lid on the staining dish and place it on a microplate shaker platform that is equipped with a non skid pad Shake for the recommended amount of time 3 Deparaffinization If using xylene work in a fume hood 30 min A Pour 200 mL of xylene into a green clearing agent dish B Transfer the rack of baked slides to the green clearing dish containing the xylene C Incubate the slides at RT for 5 min Agitate frequently by moving the rack up and down D Discard the used xylene and refill with another 200 mL of fresh xylene Incubate slides at RT for 5 min with frequent agitation E Repeat Step D above F Remove the slide rack from the xylene and wash the slides twice each time with 200 mL of 100 ethanol for 5 min with frequent agitation G Remove the slides from the rack and place them face up on a paper towel to air dry for 5 min at RT If using Histo Clear A Pour 200 mL of Histo Clear into a green clearing dish and insert an empty slide rack B Set the dry oven or hybridization system to 80 1 C C Bake the slide for 3 min to melt the paraffin D Immediately insert the warm slides into the Histo Clear and agitate frequently by moving the rack up and down for 5 min at RT E Discard the used Histo Clear and refill the dish with another 200 mL of fresh Histo Clear Agitate frequently by moving the rack up and down for another 5 min at RT F Remove the slide rack from the Histo Clear and w
63. t the specimen slide and slowly place it onto the mounting medium at an angle Make sure that the tissue comes into contact with the mounting medium first before completely letting go of the glass slide to overlap with the cover glass After mounting flip the slide over and place it on its edge on a laboratory wipe to soak up and remove excess mounting medium Allow the slide to dry at RT in the dark for 15 min Do not bake the slides to speed up the drying process To prevent bubble formation seal all 4 edges of the cover glass with a flat black colored nail polish iridescent or colored nail polish can autofluoresce and interfere with fluorescent imaging Image the results using a brightfield and or fluorescence microscope Store slides at RT 22 ViewRNA MISH Tissue 2 Plex Assay User Manual Troubleshooting Contacting Technical Support Weak or No Signals on page 24 High Background on page 25 Diffused Signals on page 26 Endogenous Alkaline Phosphatase Activity on page 26 Tissue Detachment From Slide on page 27 Poor Cell Morphology on page 27 High Non Specific Binding on Glass Slide on page 27 Pink Non Specific Background Where Paraffin Was on page 28 Hydrophobic Barrier Falls Off on page 28 Fast Red Signal for TYPE 1 Target is Weak or Different in 2 Plex Versus 1 Plex on page 28 TYPE 1 Target Signals Observed in the Channel for TYPE 6 Target on page 29 Co localized Fast Blue and Fast Red Signals When Using Only TYPE 6
64. tg lieti LE PEE xe dp 41 Important Procedural Notes 41 Modifications to Part 1 Sample Preparation and Target Probe Hybridization 42 Introduction About This Manual Assay Overview Safety Warnings and Precautions on page 2 Required Materials and Equipment on page 2 Microscopy and Imaging Equipment Guidelines on page 6 About This Manual This manual provides complete instructions for performing the ViewRNA ISH Tissue 2 Plex Assay for visualization of one or two target RNAs in formalin fixed paraffin embedded FFPE samples prepared in accordance with the guidelines provided Appendix E on page 41 provides a modified protocol for OCT embedded frozen tissue sections Assay Overview In situ hybridization ISH techniques are used to visualize DNA or localize RNAs within cells However in situ analysis of RNA in particular has always been limited by low sensitivity and complicated probe synthesis The ViewRNA ISH Tissue 2 Plex Assay based on highly specific branched DNA signal amplification technology provides robust simultaneous in situ detection of any two target mRNAs within FFPE tissue sections with single copy sensitivity Figure 1 1 shows an overview of the assay workflow Figure 1 1 ViewRNA ISH Tissue 2 Plex Assay Workflow Sample Target Signal Amplification Visualization Preparation Hybridization and Detection Target specific Probe Sets PreAmplifier
65. to give high background Prevalidate each new batch of slides by drawing a hydrophobic barrier onto an empty slide without fixed tissue allow it to dry for 20 30 min boil in pretreatment solution for 40 min to determine if the hydrophobic barrier is intact and the slides are suitable for the assay Incorrect hydrophobic pen Use the recommended Hydrophobic Barrier Pen Affymetrix QVC0500 or Vector Laboratories H4000 Hydrophobic barrier was not completely dried Be sure that the hydrophobic barrier is completely dry before proceeding to the next step This can be 20 30 min or longer depending on how heavily the barrier is created Fast Red Signal for TYPE 1 Target is Weak or Different in 2 Plex Versus 1 Plex Table 4 11 Troubleshooting Weak or Different Fast Red Signal for TYPE 1 Target in 2 Plex Versus 1 Plex Probable Cause Recommended Action Cross inhibition of LP1 AP by Fast Blue precipitate Assign lower expressing target to TYPE 6 Fast Blue and higher expressing target to TYPE 1 Fast Red Co localization of TYPE 1 and TYPE 6 targets Perform a 1 plex assay for each target Assign lower expressing target to TYPE 6 Fast Blue and higher expressing target to TYPE 1 Fast Red If co localization study is desired try reducing development time for Fast Blue from 30 min to 10 15 min Chapter 4 Troubleshooting 29 TYPE 1 Target Signals Observed in the Channel for TYP
66. uidelines on page 8 Part 1 Sample Preparation and Target Probe Hybridization Table 3 1 ViewRNA ISH Tissue 2 Plex Assay Sample Preparation and Target Probe Hybridization Step Action 1 Bake Slides A Set the dry oven or hybridization system to 60 1 C 65 min B Label the slides with a pencil C Bake the slides following the instructions below Dry oven Insert slides into the slide rack and bake slides for 60 min ThermoBrite System Keep the lid open and bake slides for 60 min Make sure that the temperature of the ThermoBrite System is validated with the lid open 2 Prepare Buffers A Verify that the hybridization system is set to 40 1 C and that it is appropriately humidified Reagents and B Prepare 3 L 1X PBS Add 300 mL 10X PBS and 2 7 L ddH O to a 3 L capacity container Equipment While c prepare 200 mL 10 NBF Work in fume hood Add 178 mL 1X PBS 22 mL 37 Slides Bake formaldehyde to a 200 mL capacity container and mix well D Prepare 4 L Wash Buffer Add the components below in the order listed to a 4 L capacity container and mix well 3L ddH O 36 mL Wash Comp 1 10 mL Wash Comp 2 Adjust the total volume to 4 L with ddH O E Prepare 500 mL 1X Pretreatment Solution Add 5 mL 100X Pretreatment Solution and 495 mL ddH 0 to a 1 L glass beaker F Prepare 200 mL Storage Buffer for optional stopping point Add 60 mL Wash Comp 2 and 140 mL ddH 0 to a 200 mL capacity conta
67. x Assay this balance between signal strength and tissue morphology is largely sample dependent tissue types as well as the modes of fixation and sample preparation and can be achieved by optimizing the pretreatment conditions to empirically determine the optimal time for heat treatment and protease digestion When optimizing the pretreatment conditions for your tissue type choose a target that is known to be expressed in the tissue of interest with medium to medium high levels of expression This will avoid possible signal saturation that may be associated with extremely high expressing targets and allow for detectable changes in the signals to be assessed as a function of the different pretreatment conditions In general a housekeeping gene with medium high expression such as GAPD or ACTB can be used for this purpose Once the optimal pretreatment conditions are determined they can generally be used for most targets within the particular tissue If the transcript is expressed at an extremely low level the optimal pretreatment condition may need to be one that favors signal over morphology Sample Pretreatment Optimization Setup Ten FFPE tissue sections from the same block are treated with different set of pretreatment conditions prior to target probe hybridization step Slide 7 serves as a no probe control while the remaining 9 slides are processed with the control target probe set Table B 1 on page 35 provides sample pretreatment conditi
68. y visible under brightfield the red dots generally have a much higher contrast than the blue dots especially in the presence of hematoxylin Thus when the detection of only one target 1 plex assay is desired we recommend using either TYPE 1 or TYPE 6 probe set and developing the signal as Fast Red See Appendix C Modified Protocols for 1 Plex Assay on page 35 for instructions on how to shorten the length of the assay when developing Fast Red or Fast Blue as a single plex When performing a 2 plex assay we recommend assigning the TYPE 1 probe set Fast Red to the more important target of the two Reserve the TYPE 6 probe set Fast Blue for the less critical target such as a housekeeping gene Due to the nature of the chromogenic assay and the sequential development of Fast Blue before Fast Red signals large quantities of blue precipitate that are deposited particularly when a TYPE 6 target is expressed homogeneously at high level have the potential to partially block subsequent hybridization of the TYPE 1 Label Probe and consequently the development of the Fast Red signal For this reason the target assigned to Fast Blue should preferably have lower expression than the one assigned to Fast Red to ensure against potential interference with Fast Red signal development downstream If only medium and high expressing housekeeping targets are available in a particular tissue type and the critical target of interest has low to medium expression a 2

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