- Advanced Cell Diagnostics
Contents
1. cccccssscccceesssseeeeeeseaes 34 Flush the instrument cccccccccccsscccseccssecessecesseceseceseeceseeeeseeeeseeceseeesseeeseees 34 Purge and prime the system with clean distilled water c ccccccssceseeees 34 Purge and prime the system with fresh reagents cccccccssccssscesseeesseeens 35 Appendix C Safety cccccssscccccssssceccceesscecceessceeceessseeeceeeesaes 37 Chemical safety 0 ccccccccccscccssccssccssccesecesescsscceseceseccseecnsecesscesseesseeseeeseeeses 37 Biological hazard safety cccccccccsccsscessccssecesecessecsseceseceseecsseesseseesesseensees 37 4 RNAscope VS Assay for the Discovery XT User Manual Red Documentation and support 06 Obtaining MSDSS cccccccsccesssceesseserseeeens Obtaining SUPPOrt ccccccccceeseceesseeesseeeens Contact information cccccccccccsesessesesseeeneens Limited product warranty ccccccssccessesenees RNAscope VS Assay for the Discovery XT User Manual Red ADVANCED CELL DIAGNOSTICS ADVANCED CELL DIAGNOSTICS Chapter 1 Product Information Before using this product read and understand the information in Appendix C Safety on page 37 in this document IMPORTANT We recommend reading the entire user manual before beginning any protocols About this guide This user manual provides two versions of the RNAscope VS Assay e Chapter 4 Semi automated RNAscope VS Assay start2. User Logins Disabled Host Ibs 9200 _ Ventana Medical Systems Ciscowary XT Staining Module Address H0 fe Download Tests Discovery XI Staining Module Select Test Function bo Rur Tex Bat Code Bloei AT Tasi Cei Conditioner xT Ted Cel Conditioner H2 WT Test Cenvershg DT Teast Decortaminate System ST Tea Cispence Reagent XT Tes Dran System AT Te Dual Ringe S50 DST Teal Emply Vacuum Trap mT Test Fhad Pisths T Tes Home Node Plae Teat Home Reagent Tray aT Ted Jel Dran AB AT Test Jet Drain SSC 7 Tex Jag Nozzle Plate DW ST Test Jog Reagent Trap CW wT Tes Fimea wT Teri Fiege CCT ST Tea Purge CE2 AT Test Purge EZ Prep ST Teg Purge LCS ST Test Pinge Option T Test gt Purge Reso Bull T jHostiNexES 10S 00 Side Tray Foston podsta 9 Double click on Test Prime XT 10 Repeat Step 9 four more times Purge and prime the system with fresh reagents Purge and prime the system with fresh reagents k Remote viz oo Reagent Tray Postion no data Times 4 19 2012 TIS PMO Run Print et i View i n Protocols q Register Discovery AT Stains Serial 713607 Unplug the yellow tubing manitold and then load the bulk reagent containers containing fresh buffers back on the instrument 2 Click Test on the main screen and then select Function Test RNAscope VS Assay for the Discovery XT User Manua
3. a tae bead i jv Baking Baking Extended Low Temperature Medium Incubation Time eo Deg C o Hr 32 Min W Deparaffinization fended Depar Extended depar allows wax to be melted in LES before regular depar with EZ prep Depar Cycle 1 8 minutes Deparalfinization High Temperature Incubation Time 65 Deg C E Minutes Cycle 1 High Temperature 75 Deg C 16 minutes Pre Fixative W Cell Conditioning Conditioner 1 W Conditioner 2 Cell Conditioner 2 Ver High Temperature 100 Deg E z Save As jv Mild CE2 Standard CC M Pretreatment 2 W Use Beschon Butter for PT2 Clear Deere osc cD DEDEDE Incubation Time and Temp for Pretreat A Edit Filter Print A Pretreatment 2 Temp AE Low Temperature Incubation Time 38 Deg C 12 Minutes Close W Pretreatment 3 W Use Reaction Buffer for PTS W Heat slides for PT3 AB Incubation Time and Temp for Pretreat B Pretreatment 3 Temp RB Low Temperature Incubation Time 38 Deg C iz Minutes Stre fie Oe Print PROBE 1 0761 Hybridization Low Temperature Clear as Deg C Edit Filter Amp 1 incubation temperature Detection 1 Temp Low Temperature 55 Deg C Amp 3 incubation temperature Detection 3 Temp Low Temperature 55 Deg C Close Erg px Pees ara Z Specify probe hybridization time and temperature RNAscope VS Assay for the Di
4. 1 3 dots cell visible at 20 40X magnification 2 4 10 dots cell No or very few dot clusters visible at 20 40X magnification 3 gt 10 dots cell Less than 10 positive cells have dot clusters visible at 20X magnification 4 gt 10 dots cell More than 10 positive cells have dot clusters visible at 20X magnification Discount cells with artificially high nuclear background staining Quantitative Image Analysis RNAscope Spot Studio Software is designed for pathologists with no prior training in image analysis This intuitive software allows users to get statistical results with complete information of cell count region and number of spots cell Simply load any image select a region of interest define settings and run analysis followed by a quality control review before results are exported Further information is available on our website at www acdbio com Troubleshooting RNAscope VS Assay for the Discovery XT User Manual Red 31 ASD ADVANCED CELL DIAGNOSTICS For troubleshooting information please contact technical support at support acdbio com Control example If the assay is successful the staining should look like the following images Figure 2 RNAscope VS Assay detection of HPV E6 E7 mRNA in cervical cancer FFPE tissue 32 RNAscope VS Assay for the Discovery XT User Manual Red ADVANCED CELL DIAGNOSTICS Appendix A Recommended Guidelines We highly recommend following the guidelines for
5. 234 Component Storage Generic dispensers Cat No 771 741 and 771 742 Component Storage Counterstain 1 dispenser fill dispenser with Hematoxylin Room temperature 20 25 C Counterstain 2 dispenser fill dispenser with bluing reagent Room temperature 20 25 C mRNA Red Amplification amp Pretreatment PTO Kit Cat No 760 235 Component Storage mRNA Red Detection Kit Cat No 760 234 A C mRNA Red Amplification Kit Cat No 760 236 Room temperature 20 25 C mRNA Red Pretreatment Kit Cat No 760 237 Room temperature 20 25 C RNAscope VS Assay for the Discovery XT User Manual Red 9 ADVANCED CELL DIAGNOSTICS Equipment and buffers Fa Component DISCOVERY XT automated slide stainer EZPrep Buffer LCS Buffer RiboWash Buffer RiboCC Buffer used for automated cell conditioning CC2 Reaction Buffer DISCOVERY 1X SSC Wash in Option Bottle User supplied materials F DISXT 750000 950 100 650 010 760 105 760 107 950 300 950 210 Cat No IMPORTANT Do not substitute other materials for the SuperFrost Plus Slides listed in the following table r Description Supplier Cat No SuperFrost Plus Slides required Fisher Scientific 12 550 15 100 ethanol EtOH American Master Tech Scientific MLS ALREAGAL Xylene Fisher Scientific MLS X3P 1GAL 10 neutral buffered formalin NBF MLS Paraffi
6. Cell Conditioning Pretreat 2 Pretreatment 2 Pretreat A and Pretreatment 3 Pretreat B conditions for e Any new or previously untested FFPE tissue types e Samples prepared differently than the protocol in Chapter 3 Prepare and Pretreat Samples on page 12 1 Stain six representative slides using the positive and negative control probes according to the following table Slide No Probe Manual Cell Pretreatment 2 Pretreatment 3 Conditioning 1 Positive control 10 MIN 12 MIN 12 MIN 2 Negative control 10 MIN 12 MIN 12 MIN 3 Positive control 15 MIN 12 MIN 12 MIN 4 Negative control 15 MIN 12 MIN 12 MIN 5 Positive control 30 MIN 12 MIN 12 MIN 6 Negative control 30 MIN 12 MIN 12 MIN Evaluate staining and tissue morphology as in Chapter 6 Evaluate the results on page 31 and determine which pretreatment condition yielded the highest positive control signal and lowest negative control signal Positive control signal should have a staining score of 3 or higher and the negative control signal should be O Use the optimized pretreatment conditions to run the assay with the target probe If none of the conditions are satisfactory contact technical support at support acdbio com RNAscope VS Assay for the Discovery XT User Manual Red 33 ADVANCED CELL DIAGNOSTICS Appendix B Maintain the Instrument If you have not used your DISCOVERY XT for one week or longer bacterial growth and or
7. Pretreatment 3 Pretreat B Brain and spinal cord 12 MIN 12 MIN Breast cancer 12 MIN 12 MIN Cell lines 12 MIN 12 MIN Colon 12 MIN 12 MIN Gl tract 12 MIN 12 MIN Head and neck cancer 12 MIN 12 MIN Heart 12 MIN 12 MIN Kidney 12 MIN 12 MIN Liver 12 MIN 12 MIN Lung 12 MIN 12 MIN Lymphoma 8 MIN 8 MIN Placenta 12 MIN 12 MIN Prostate 12 MIN 12 MIN Skin 12 MIN 12 MIN Stomach 12 MIN 12 MIN Thymus 8 MIN 8 MIN Tonsil 8 MIN 8 MIN Xenograft derived from 12 MIN 12 MIN cell lines Xenograft derived from 12 MIN 12 MIN primary tumor RNAscope VS Assay for the Discovery XT User Manual Red ADVANCED CELL DIAGNOSTICS Click Save As then select a protocol number from the drop down menu and choose a protocol name for each probe Click Save Click Close to go back to the main screen Assign a probe number from the list to each probe of interest For each probe selected assign a protocol Print the labels 1 Select the Print Label icon trom the bottom of the home page screen Select your preferred template or create a new template To create a new template refer to the Ventana System User Manual tor details Select the protocol you created for the RNAscope VS Assay Click on Protocol to add and print the label RNAscope VS Assay for the Discovery XT User Manual Red 19 ADVANCED CELL DIAGNOSTICS Manually pretreat the samples Materials required Materials provided by the RNAscope VS Other materials and equipm
8. Repeat steps 2 and 3 for each slide 6 Air dry slides for 5 MIN Proceed to Chapter 6 Evaluate the results on page 31 30 RNAscope VS Assay for the Discovery XT User Manual Red ADVANCED CELL DIAGNOSTICS Chapter 6 Evaluate the results Examine tissue sections under a standard bright field microscope at 20 40X magnification e Assess tissue and cell morphology e Assess positive control signal strength Positive control signal should be visible as punctate dots within cell nuclei at 20 40X magnification e Assess negative control background One dot to every 10 cells displaying background staining per 20X microscope field is acceptable e Evaluate target probe signal using the scoring guidelines in the next section Scoring guidelines The RNAscope Assay enables a semi quantitative scoring guideline utilizing the estimated number of punctate dots present within each cell boundary An example of how to develop such a guideline for semi quantitative assessment of RNAscope staining intensity is presented below for a gene with expression level varying between 1 to gt 10 copies per cell Note f your gene expression level is higher or lower than this range you may need fo scale the criteria accordingly Categorize staining into five grades 0 1 2 3 and 4 according to the following table Staining Microscope objective scoring score O No staining or less than 1 dot cell 40X magnification
9. at www who int csr resources publications biosafety who cds csr_lyo 2004 11 en e Information about the Registration Evaluation Authorisation and Restriction of Chemicals REACH can be found at eur lex europa eu LexUriServ LexUriServ do uri 0J L 2010 133 0001 0043 EN PDF 38 RNAscope VS Assay for the Discovery XT User Manual Red ADVANCED CELL DIAGNOSTICS Documentation and support Obtaining MSDSs Material Safety Data Sheets MSDSs are available at www acdbio com product_literature html For the MSDSs of chemicals not distributed by Advanced Cell Diagnostics contact the chemical manufacturer Obtaining support For the latest services and support information go to www acdbio com product_support html At the website you can e Access telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support e Search for user documents MSDSs application notes citations training videos and other product support documents e Find out information about customer training events Contact information Advanced Cell Diagnostics Inc 3960 Point Eden Way Hayward CA 94545 Toll Free 1 877 576 3636 Direct 1 510 576 8800 Fax 1 510 576 8801 Information info acdbio com Orders orders acdbio com Support Email support acdbio com Limited product warranty Advanced Cell Diagnostics Inc and or its attil
10. pads 2 Cover each slide with distilled water Note Avoid pouring distilled water directly on the sections Loading the reagents Start the run 22 1 Remove the nozzle caps of the filled dispensers and place them on their holders It needed remove any air bubbles at the nozzle tips by squeezing out one drop of reagent Load dispensers onto the reagent racks Remove the yellow locking ring trom the dispensers in the prefilled mRNA RED Detection Kit Refer to the instructions provided by Ventana Medical Systems load the reagent racks onto the reagent carousel 1 Click the Run button 2 Follow the instructions on the instrument screen Select the Reagent Reagent Tray Loaded and Reagent Caps Removed check boxes 3 Enter the number of slides Click the Running button Automated assay will finish in 8 HRS IMPORTANT Before leaving the instrument unattended ensure all reagents and slides are successtully registered and the instrument is running RNAscope VS Assay for the Discovery XT User Manual Red ADVANCED CELL DIAGNOSTICS Complete the run 1 After the run is complete place nozzle caps back on the dispensers Store reagent racks at 4 C until next use Wash the slides 1 Add 200 ml diluted detergent to a Tissue Tek Staining Dish Submerge a Tissue Tek Slide Rack into the staining dish Open the instrument slide tray and unload slides Decant solution on the slides into the slide tray then immedi
11. 0 EtOH Incubate the slides in 100 EtOH for 1 MIN at RT with agitation Repeat Step 6 with fresh 100 EtOH Remove the slides from the rack and place on absorbent paper with the section face up Air dry for 5 MIN at RT While slides are drying place printed labels on the slides 20 RNAscope VS Assay for the Discovery XT User Manual Red ADVANCED CELL DIAGNOSTICS IMPORTANT Labels must be in place prior to the next section Insert the slides into a Tissue Tek Slide Rack and proceed to the next section Pretreat the slides Begin heating 1X Pretreat 2 while FFPE slides are baking at 60 C or during the previous section IMPORTANT Do not boil 1X Pretreat 2 more than 30 MIN before use 1 Heat 1X Pretreat 2 to 98 104 C a Place the beaker containing 1X Pretreat 2 on the hot plate Cover the beaker with foil and turn the hot plate on high for 10 15 MIN b Once 1X Pretreat 2 reaches a slow boil 98 104 C turn the hot plate to a lower setting to maintain the correct temperature Check the temperature with a thermometer With a pair of forceps very slowly submerge the slide rack containing the slides into the boiling 1X Pretreat 2 solution Cover the beaker with foil and boil the slides for the amount of time specitied in the following table Tissue type Treatment time Brain and spinal cord 15 MIN Breast cancer 15 MIN Cell lines 10 MIN Colon 15 MI
12. 12 Dehydrate embed and cut the sample ccccccccssecessseeesseeesssesesseeeens 12 Chapter 4 Semi automated RNAscope VS Assay ccccceeeeeees 14 NV TIO sop n sein insite AA 14 Prepare the MALE AS eijcscscosnarasqieriarcmnennarnaernie emia 15 Materials regUired o22acecsaaensasecaasesaaassanniaanniianeaagnaaaxiaaasananesansnaasariannaaatie 15 Prepare the instrument ccccccccccssccessccessccesecccsseccsecesseeessesesseeesseeesseenss 15 Prepare instrument reagents ccccseeeeeeeecceeeeeaeeeeeeeeceeeeeaaeeeeeeeeeeeeeaaas 15 AK s o k wel 1 1 0 1 eee ne enn ewe ee Te 16 Prepare deparaffinization reagent cccccccssccessccesscceseccesecessesesseeenseeens 16 Prepare mounting recagent cccccccsseeeeeeeccceceeeeeeeeeeeeeeeeeeaaueeeeeeeeeeeeaaas 16 Prepare 1X Pretreat 2 ornan 16 Create an instrument protocol cccccccsccesscceseceeseeessecesseeessesesseeeseeeeseees 16 SELCA elo ae een ee eee 19 Manually pretreat the samples cccccccccccssccesssecesseeeessecesseecesseeeessesenseeeens 20 Moaterials TEQWIRCO c lt casessacaccancaancdaantsaaseasdunsaaacetaateaanessaaneeansaaneasanbsnabeananndies 20 Bake the slides cccccccscccssccsssccssecessecessecessscesecessecesseceseecseeeseeenseeees 20 Deparaffinize FFPE sections c ccccccccsscessssessecessecesseceseecesesessesesseeenseeens 20 RNAscope VS Assay for the Discovery XT User Manual Red ADVANCED CELL
13. 8 HRS IMPORTANT Before leaving the instrument unattended ensure all reagents and slides are successtully registered and the instrument is running Complete the run 1 After the run is complete place nozzle caps back on the dispensers Store reagent racks at 4 C until next use RNAscope VS Assay for the Discovery XT User Manual Red 29 ADVANCED CELL DIAGNOSTICS Wash the slides 1 Add 200 ml diluted detergent to a Tissue Tek Staining Dish Submerge a Tissue Tek Slide Rack into the staining dish Open the instrument slide tray and unload slides Decant solution on the slides into the slide tray then immediately load slides into the Tissue Tek Slide Rack submerged in detergent Rinse oil off the slides by moving the slide rack up and down in the dish 10 times Replace the detergent with distilled water and rinse slides by moving the slide rack up and down 10 times Repeat Step 6 3 5 times Mount the samples 1 Remove the slide rack from the staining dish and dry slides in a 60 C dry oven for 15 MIN IMPORTANT The RED substrate is alcohol sensitive Do not dehydrate the slides in alcohol 2 Cool the slides for 5 MIN at RT 3 Briefly dip one slide into into fresh pure xylene and immediately place 1 2 drops of EcoMount on the slide before the xylene dries WARNING Use the EcoMount mounting medium only 4 Carefully place 24 mm x 50 mm coverslip over the tissue section Avoid trapping air bubbles 5
14. DIAGNOSTICS Pretreat the slides ccccccccccssccsssccssscessecessecessecessecessecessecesseceseeecseeesseeess 21 Run the RNAscope VS Assay ccccccccsssccsscccssccesecessecessecessecesseeeeeeeeeeeseees 22 Materials required cccccccccccssccesscceseccssecessecessecesseccssecesseceseeeeseeenseeeeees 22 Loading the slides cccccccsscccsssceesseccessecesseecesseecesseceesesesseesesseeeesseeens 22 Loading the reagent cccccccscccsssccesseccessecesseecesseecessecceseecesseesenseeeeseeess 22 SPI PNG PUI EENE EE EN E 22 Complete the run ccccccccccccssccesscccesseccsseecesseecessesesseesesseecessesesseesesseeeeseess 23 Wash ine slidas serspississoprepsuirs sirinin niinniin ER SR i SERS RAS ERa 23 Mount the samples ccccccccccsscccssccssscessecessecessecessecesseceseeceseeeeseseeseeeeees 23 Chapter 5 Fully Automated RNAscope VS Assay ccccceeeeeees 23 NN FICTION oaa EN 24 Prepare the materials ccccccsssccessscesssecesseccessecesseecesseccsssecenseeeenseeeseeess 25 Materials required ccccccccccsscccssccssecessecessecessecessecesseccsseesseeessesenseeeseees 25 Prepare the instrument ccccccscccsscccssccesscceseeccsseeeseceeseccsseeesseeesseeesseeess 25 Prepare instrument reagent cccceseeeeeeeecceceeaeeeeeeeeeeeeeeeaueeseeeeeeeeenaas 25 Prepare detergent cccccccsscccsssceesseccessecesseesesseecesseccessesesssesessesenseeeens 26 Prepare mount
15. L STOPPING POINT Use sectioned tissue within 3 months Store sections with dessicants at RT RNAscope VS Assay for the Discovery XT User Manual Red 13 ADVANCED CELL DIAGNOSTICS A Chapter 4 Semi automated RNAscope VS Assay Most sample types require manual pretreatment prior to running the automated RNAscope VS Assay For brain and spinal cord sections you may fully automate manual pretreatment steps See Chapter 5 Fully Automated RNAscope VS Assay on page 23 Workflow Prepare the materials 4 Bake the slides 30 MIN 4 Deparaffinize FFPE tissue 20 MIN 4 Pretreat the slides 20 45 MIN 4 Run the RNASCOPE VS Assay 8 HRS RNAscope VS Assay for the Discovery XT User Manual Red Prepare the materials ADVANCED CELL DIAGNOSTICS Materials can be prepared ahead of time or while baking the slides unless otherwise stated See Bake the slides on page 20 Materials required Materials provided by Advanced Cell Diagnostics Materials provided by Ventana Medical Systems Other materials and equipment VS Target Probe e VS Positive Control Probe e VS Negative Control Probe e 10X Pretreat 2 e VS Pretreat A Red e VS Pretreat B Red e VS Amp 1 Red e VS Amp 2 Red e VS Amp 3 Red e VS Amp 4 Red e VS Amp 5 Red e VS Amp 6 Red e VS Amp 7 Red e VS Hematoxylin VS Bluing reagent Prepare the instrument DISCOVERY XT slide stainer EZ
16. N Gl tract 15 MIN Head and neck cancer 15 MIN Heart 15 MIN Kidney 15 MIN Liver 30 MIN Lung 15 MIN Lymphoma 10 MIN Placenta 15 MIN Prostate 15 MIN Skin 15 MIN Stomach 15 MIN Thymus 10 MIN Tonsil 10 MIN Xenograft derived from cell lines 7 MIN Xenograft derived from primary tumor 15 MIN This procedure can be automated for these tissue types See page 23 Use the forceps to immediately transfer the hot slide rack from the 1X Pretreat 2 to the staining dish containing distilled water Do not let the slides cool in Pretreat 2 Wash slides 3 5 times by moving the Tissue Tek Slide Rack up and down in the distilled water Repeat Step 4 with fresh distilled water RNAscope VS Assay for the Discovery XT User Manual Red 21 ADVANCED CELL DIAGNOSTICS Directly proceed to Loading the slides into slide holders on the instrument Run the RNAscope VS Assay Materials required e Prepared slides e Prepared instrument reagents e Distilled water e Dawn detergent e Fume hood e Xylene Tissue Tek Staining Dish 1 Tissue Tek Clearing Agent Dish xylene resistant 1 Tissue Tek Vertical 24 Slide Rack e EcoMount e Cover Glass 24 mm x 50 mm Loading the slides IMPORTANT Prior to loading the slides ensure heater pads are completely dry Wipe off any solution using laboratory tissue paper 1 Load each slide onto a heater pad with the label facing away from you Ensure that the slides sit securely on the
17. Prep Buffer LCS Buffer RiboWash Buffer Reaction Buffer DISCOVERY 1X SSC Option Bottle Probe dispensers mRNA Pretreatment Kit mRNA Probe Amplification Kit mRNA Red Detection Kit Generic dispensers Distilled water Glass beaker 1 or 2 L Hot plate Dawn detergent Fume hood Xylene 100 ethanol EtOH Tissue Tek Staining Dish 3 Tissue Tek Clearing Agent Dish xylene resistant 3 Tissue Tek Vertical 24 Slide Rack EcoMount Cover Glass 24 mm x 50 mm e If the instrument has not been used for 21 week follow the guidelines for instrument maintenance in Appendix B on page 34 e f your instrument has been used recently run the Prime XT protocol two times to clear the fluid lines before setting up the experiment Refer to the Ventana System User Manual and Appendix B on page 34 for details Prepare instrument reagents Register new reagent kits using the wand that comes with the instrument 1 In reverse order trom AMP 7 to AMP 1 transfer the entire volume of each RNAscope VS FFPE Reagent Kit component into the correspondingly labeled dispenser 2 Transfer the rest of the RNAscope VS Reagents to the correspondingly labeled dispensers IMPORTANT Avoid cross contamination between reagents 3 Follow the dispenser product insert instructions to properly prime and handle the dispensers 4 Press the dispenser caps down tightly Note Store tightly capped dispensers at 4 when not in u
18. Reagent Cat No Content Quantity Storage RNAscope VS Positive Control Probe Various Probe targeting common 7 mL x 1 bottle 4 C species PPIB housekeeping gene RNAscope VS Negative Control Probe 310043 Probe targeting bacterial 7 mL x 1 bottle 4 C DapB gene dapB RNAscope Control Slides The RNAscope Control Slides Cat No 310045 for Human control slide Hela Catalog No 310023 tor Mouse control slide 313 contain FFPE cell pellets sectioned and mounted on slides The control slides can be used for assay control with the RNAscope Positive Control Probe and RNAscope Negative Control Probes The slides have a shelf lite of 6 months trom the shipment date when stored at 2 8 C with dessicants RNAscope VS Assay for the Discovery XT User Manual Red 7 ADVANCED CELL DIAGNOSTICS RNAscope VS Reagents The RNAscope VS Reagents contain all the reagents needed to run the RNAscope assay on the Ventana Discovery ULTRA except for the RNAspecific probes The contents of the RNAscope VS Reagents consist of the RNAscope VS Reagent Kit RED Cat No 320610 the RNAscope VS Accessory Kit Cat No 320630 and the RNAscope Pretreat 2 Pack Cat No 320043 The kits provide enough reagents to stain 6O standard slides The reagents have a shelf lite of six months from the shipment date when stored as indicated in the following table RNAscope VS Reagent Kit RED Cat No 320610 Reagent Quanti
19. USER MANUAL ADVANCED CELL DIAGNOSTICS RNAscope VS Assay For DISCOVERY XT RED Document Number 320523 For Research Use Only RUO Rev Date 20150318 For Reseach Use Only RUO Trademarks RNAscope is a registered trademarks of Advanced Cell Diagnostics Inc VENTANA and DISCOVERY are trademarks of Roche All other trademarks belong to their respective owners Citing RNAscope VS in Publications When describing a procedure for publication using this product please refer to it as the RNAscope VS Assay and cite Wang F Flanagan J Su N Wang LC Bui S Nielson A Wu X Vo H T Ma XJ and Luo Y RNAscope A Novel n Situ RNA Analysis Platform for Formalin Fixed Parattin Embedded Tissues J Mol Diagnostics 2012 14 22 29 Disclaimers Advanced Cell Diagnostics 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 Advanced Cell Diagnostics Inc assumes no liability for any errors omissions or for any damages resulting trom the use of this information Copyright 2015 Advanced Cell Diagnostics Inc All rights reserved ADVANCED CELL DIAGNOSTICS Contents Chapter 1 Product Information ccccsssccccessssseccceesssseeceessseeeeens 6 About this QUICG ci 202a s esccscaceicsacsrascsnsacsvaicsaeciseaesataiaieassaieie
20. ately load slides into the Tissue Tek Slide Rack submerged in detergent Rinse oil otf the slides by moving the slide rack up and down in the dish 10 times Replace the detergent with distilled water and rinse slides by moving the slide rack up and down 10 times Repeat Step 6 3 5 times Mount the samples 1 Remove the slide rack from the staining dish and dry slides in a 60 C dry oven for 15 MIN IMPORTANT The RED substrate is alcohol sensitive Do not dehydrate the slides in alcohol 2 Cool the slides for 5 MIN at RT 3 Briefly dip one slide into into fresh pure xylene and immediately place 1 2 drops of EcoMount on the slide before the xylene dries WARNING Use the EcoMount mounting medium only 4 Carefully place 24 mm x 50 mm coverslip over the tissue section Avoid trapping air bubbles 5 Repeat steps 2 and 3 for each slide 6 Air dry slides for 5 MIN 7 Proceed to Chapter 6 Evaluate the results on page 31 Chapter 5 Fully Automated RNAscope VS Assay Use this protocol for brain and spinal cord sections The Fully Automated RNAscope VS Assay shares many of the same steps as the Semi automated RNAscope VS Assay Do not perform the manual pretreatment steps RNAscope VS Assay for the Discovery XT User Manual Red 23 ADVANCED CELL DIAGNOSTICS IMPORTANT We strongly recommend you run the FFPE RNAscope Control Slides Cat No 310045 using the positive and negative control probes along with you
21. e Appendix C Safety Chemical safety N WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions e Read and understand the Material Safety Data Sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain MSDSs see Documentation and support in this document e Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example satety glasses gloves or protective clothing e Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood e Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory e Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations e IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Biological hazard safety WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit intectious di
22. e a Ta gt z T in a r 1 Tissue section 2 Hybridize to target RNA 3 Amplify signal Few sor lt gt Start with properly prepared Hybridize gene specific probe Probes are hybridized to a cascade of signal Visualize target sections and pretreat to allow pairs to the target mRNA amplification molecules culminating in binding of RNA using a access to target RNA HRP or AP labeled probes The 2 0 Assay standard bright enhances signal further with additional field microscope amplification steps Add DAB or Fast Red substrate to detect target RNA Kit contents and storage The RNAscope VS Assay requires the RNAscope VS Probes and the RNAscope VS Reagents available trom Advanced Cell Diagnostics RNAscope VS Probes The RNAscope VS Probes consist of the user specified Target Probe and the Positive and Negative Control Probes Visit www acdbio com products target probes search product to tind a gene specific Target Probe Visit www acdbio com products targetprobes controls housekeeping to order appropriate Control Probes Each probe is sufficient for staining 30 standard slides The probes have a shelf life of six months trom the shipment date when stored as indicated in the following table Target Probes w Reagent Cat No Content Quantity Storage RNAscope VS Target Probe species Various Probe targeting specific RNA 7 mL x 1 bottle 4 C gene Control Probes
23. ent Reagents e 10X Pretreat 2 e Drying oven e FFPE slides e Tissue Tek Vertical 24 Slide Rack e Distilled water e Fume hood e Xylene e 100 ethanol EtOH e Tissue Tek Clearing Agent Dish 2 e Tissue Tek Staining Dish 2 e Glass beaker 1 or 2 L e Hot plate Bake the slides 1 Bake slides in a dry oven for 30 MIN at 60 C STOPPING POINT Use immediately or store at RT with dessicants tor 1 week Prolonged storage may degrade sample RNA If you continue prepare the materials for the following protocols while the slides are baking Deparaffinize FFPE sections in the next section Pretreat the slides on page 21 and Run the RNAscope VS Assay on page 23 See Prepare the materials on page 15 Deparaftinize FFPE sections IMPORTANT If you have not done so already create a protocol for your instrument and print slide labels during this procedure See pages 16 19 1 Place slides in a Tissue Tek Slide Rack and submerge in the first xylene containing clearing agent dish in the fume hood Incubate the slides in xylene for 5 MIN at RT Agitate the slides by occasionally lifting the slide rack up and down in the clearing agent dish Remove the slide rack trom the first xylene containing dish and immediately place in the second xylene containing clearing agent dish in the tume hood Repeat Step 2 Remove the slide rack trom the second xylene containing dish and immediately place in the staining dish containing 10
24. for the Discovery XT User Manual Red Materials required ADVANCED CELL DIAGNOSTICS e Prepared instrument reagents e Distilled water e Dawn detergent e Fume hood e Xylene e Tissue Tek Staining Dish 1 e Tissue Tek Clearing Agent Dish xylene resistant 1 e Tissue Tek Vertical 24 Slide Rack e EcoMount e Cover Glass 24 mm x 50 mm Loading the slides IMPORTANT Prior to loading the slides ensure heater pads are completely dry Wipe off any solution using laboratory tissue paper Loading the reagents Start the run 1 Load each slide onto a heater pad with the label facing away from you Ensure that the slides sit securely on the pads 2 Cover each slide with distilled water Note Avoid pouring distilled water directly on the sections 1 Remove the nozzle caps of the filled dispensers and place them on their holders It needed remove any air bubbles at the nozzle tips by squeezing out one drop of reagent Load dispensers onto the reagent racks Remove the yellow locking ring trom the dispensers in the pretilled mRNA RED Detection Kit Refer to the instructions provided by Ventana Medical Systems load the reagent racks onto the reagent carousel 1 Click the Run button Follow the instructions on the instrument screen Select the Reagent Reagent Tray Loaded and Reagent Caps Removed check boxes Enter the number of slides Click the Running button Automated assay will finish in
25. gents Note In a fume hood fill a clearing agent dish with 200 ml fresh xylene Create an instrument protocol 26 a Deparaffinization T Pretnaive Cell Conditioning Ensure all containers remain covered 1 Open the NexES software and click on the Protocol button 2 Click on Create Edit Protocols go to the Procedure drop down menu and select mRNA Red DXT 2 0 3 Main protocol steps appear as shown NexES Protocol Editor Discovery XT Staining Module C Number T Wet Slide Load I List Only Registered Products Procedure mRNA Red DXT 2 0 Filter Procedures l Baking l Pretreatment 2 Pretreatment 3 Specify probe hybridization time and temperature Probe Hybridization Low Temperature Amp 1 incubation temperature Detection 1 Temp Low Temperature Amp 3 incubation temperature Detection 3 Temp Low Temperature Edit Filter M Close 4 After selecting the main protocol steps drop down menus become available Select the appropriate protocol steps by clicking on the associated check boxes as shown RNAscope VS Assay for the Discovery XT User Manual Red ALD tA ADVANCED CELL DIAGNOSTICS NexES Protocol Editor Discovery XT Staining Module A Procedure tPA l List Only Registered Products Name mRNA Red DXT 2 0 Red Test I Filter Procedures Mumber 50
26. iate s warrant their products as set forth in the ADC General Terms and Conditions of Sale found on the ADC website at www acdbio com product_support html f you have any questions please contact Advanced Cell Diagnostics at www acdbio com product_support html RNAscope VS Assay for the Discovery XT User Manual Red 39 Headquarters 3960 Point Eden Way Hayward CA 94545 Phone 1 510 576 8800 Toll Free 1 877 576 3636 For support email support acdhbio com www acdbio com ADVANCED CELL DIAGNOSTICS
27. ing on page 14 e Chapter 5 Fully Automated RNAscope VS Assay starting on page 23 Product description Background The RNAscope Assays use a novel and proprietary method of n situ hybridization ISH to visualize single RNA molecules per cell in formalin tixed parattineembedded FFPE tissue mounted on slides The assays are based on Advanced Cell Diagnostic s patented signal amplification and background suppression technology and can detect RNA molecules in archival samples and partially degraded specimens The RNAscope VS Assay allows users fo automate the highly sensitive RNAscope Assay using the Ventana DISCOVERY XT or ULTRA systems Overview The RNAscope VS Assay procedure is illustrated in Figure 1 on page 6 and can be completed on the instrument in 8 10 hours Starting with properly prepared samples sections are first pretreated and then RNA specific probes are hybridized to target RNA The signal is amplified using multiple steps followed by hybridization to horseradish peroxidase HRP or alkaline phosphatase AP labeled probes and detection using a chromogenic substrate Each single RNA transcript appears as a distinct dot of chromogen precipitate visible using a common brighttield microscope 6 RNAscope VS Assay for the Discovery XT User Manual Red ASD ADVANCED CELL DIAGNOSTICS Figure 1 Procedure overview 3 rm Oo ZZ Target RNA Specific Oligo Probes PreAMP AMP Label Probe Zz
28. ing recagent cccccccsseeeeeeecceeceeeeeeeeeeeeeeeeeeaaeeeeeeeeeeeenaas 26 Create an instrument protocol cccccccssccessccessceesecessecesseeesseeesseeesseeeseees 26 Print ihe laDelS eee eee eee S en 28 Run the RNAscope VS Assay ccccscccsscccssccessecesscessecessecessecesseceseeeseeeeseees 28 Materials required ccccccccccssccessccssecessecessecesseceseeceseeceseeeeseeeeseeenseeeeees 29 Loading the slides cccccccssccesssceesseccessecesseecesseecesseccessesesseesesseeeeseeeens 29 Loading the reagent ccccccsscccssscsesseccessecesseecesseeceseccessesesseesesseeeesseeens 29 Start the run cccccccccccscccssccessecessecesseceseeceseceeseeceseecesseceseeesseeesseseeeeeseess 29 Complete the run set asec tacts ened asta soreness 29 Wash the slides ccccccccccsssccsscccssecesecceseeceseecessecessecssecesesesecesseeeaeenaes 30 Mount the samples 0 cccccccccssccesseccessecesseecesseseessesesseesessesesseesesseeseseees 30 Chapter 6 Evaluate the results ccccccsssssscceccceesssseeeceeeesenes 31 Scoring COILS NSS oann EARRAN 31 Quantitative Image Analysis ccccccccscccessccesseccesseeeeseesesseecessesesuesenseens 31 Troubleshooting cccccccsscccsssccessecesssecesseecessecesseceessesesseesessescesesessesenseeeens 31 GGUS EE E E E 32 Appendix A Recommended Guidelines cccs cccccsssssececeessees 33 Appendix B Maintain the Instrument
29. l Red ADVANCED CELL DIAGNOSTICS Ventana Medical Systems Discovery XT Staining Module D Addiers no Addes l View Protocols Staining Module Messages i Register Users Logins Disabled Hosti NexES W105 Remote w10 22 Discovery XT Stalne Serial 1 713607 Host 10 3200 Side Tray Position no data Reagent Tray Position no data Tune 04 19 2012 7 18 PM 3 Double click on Test Purge All XT Download Tosia Discovery X1 Staining Module S Select Test Function to Purr Bae Code Elivo AT Lal ondina HI AT l Cell Conditiona B2 AT ai Coyerdip DAT Decontaminate Syster T t Daperce Reagent XT ey Dian System ST i Dual Ringe 550 ENT Emphy Vacuum Trap sT A Fhad Paths ST Home Nozze Plate Home Reagent Trap iT 4 det Drain RB ST eel Jel Drain SSC 87 dog Nozzle Plate Cal eT dog Aeagent Trap Cy xT Purge All ST Piege COT AT t Pige CEZAT a Purge EZ Piep iT Purge LOST Fiege Option 7 t Fige Rear Bult XT Bie m m User Logins Disabled Host NexES v10 5 Remote viO SE Discovery XT Stains Serial 713607 Hot O 200 in Tray Paoa deta Reagent Tray Poston podata Trom ON LAJ201Z TiS PM LOOO Repeat Step 3 Click Test on the main screen and then select Function Test Double click on Test Prime XT NODD A Repeat Step 36 RNAscope VS Assay for the Discovery XT User Manual Red ADVANCED CELL DIAGNOSTICS
30. n wax MLS gt 1X PBS MLS Microtome MLS Drying oven capable of holding temperature at 60 MLS 1 CC Water bath or incubator capable of holding MLS temperature at 40 1 C C EcoMount Biocare EM897L Tissue Tek Vertical 24 Slide Rack American Master Tech Scientific MLS LWSRA24 Tissue Tek Staining Dish 3 required American Master Tech Scientific MLS LWT4457EA ioe Clearing Agent Dish xylene resistant 3 American Master Tech Scientific MLS IWTA456EA Cover Glass 24 x 50 mm Fisher Scientific MLS 12 545 F Distilled water MLS Dawn detergent MLS Fume hood MLS Glass beaker 1 or 2 L MLS Hot plate Fisher Scientific MLS 1 1 300 49SHP Major Laboratory Supplier in North America For other regions please check Catalog Numbers with your local lab supplier 10 RNAscope VS Assay for the Discovery XT User Manual Red ADVANCED CELL DIAGNOSTICS Chapter 2 Before You Begin Prior to running the RNAscope VS Assay on your samples for the first time we recommend that you e Be familiar with the Ventana DISCOVERY XT system Refer to the Ventana System User Manual e View the video demonstrations available at www acdbio com technical support online training videos e Run the assay on FFPE RNAscope Control Slides Cat No 310045 for Human control slide Hela Catalog No 310023 for Mouse control slide 373 using the Positive and Negative Control Probes Important procedu
31. neaceacesenustontinctaees 6 Product description cccccccccssccessscsesseccessecesseceesseccessesesseesessesessseeseesenseesens 6 Borel lt o colt 2 c kan ee nn nen Tern ae ere nee ree eer eee 6 NENON RARS RNEER NNNSN RNN 6 Kit contents and stOrdge cccccccccssccesssccessecesseccesseecessesesseesesseecesseserseesesseeens 7 RNAscope VS PrODESs i isasiosstanciansdnsaneasanasanasieashancianninsnsnansensanantuasanaiannnoss 7 RNAscope Control Slides ccccccccssccesssecesseecessecesseesesseeceseeessaesesseesenaes 7 RNAscope VS Reagent ccccccccssscccesssccessseccesseeecessseeeesseecesaeesesseeeeenaes 8 Required materials from Ventana Medical Systems cccccescceesseesseeesseeens 9 Equipment and buffers cccccccccssccssccesscessccsseeeseceseccseecseseseeesseseseeesaeens 10 User supplied materials cccccccccscccseccsscccsscccsscceseccsseeessecesseeesseeesseeess 10 Chapter 2 Before You Begin ccssccccsssecsessecceesseeeseseeeessseeeeeas 11 Important procedural guidelines ccccccccccssccessccssecessecessecessecesseeeseeeeseees 11 Chapter 3 Prepare and Pretreat Samples c ccccssccsessseeeess 12 Prepare FFPE SECHONS esncsnsceasacersandeasctuadaeareaacsosetuacearsancnecetuactearsaacnosebusaanaes 12 Materials regUi red ssisrsri riri rins treri rnrn ES S EERE 12 Fix the sample ccccccsccccsssccesseccessecesssecesseccessecceeecesseecesseecesseeenseeeess
32. nstrument has been used recently run the Prime XT protocol two times to clear the fluid lines before setting up the experiment Refer to the Ventana System User Manual and Appendix B for details Prepare instrument reagents Register new reagent kits using the wand that comes with the instrument 1 In reverse order trom AMP 7 to AMP 1 transfer the entire volume of each RNAscope VS FFPE Reagent Kit component into the correspondingly labeled dispenser Transter the rest of the RNAscope VS Reagents to the correspondingly labeled dispensers IMPORTANT Avoid cross contamination between reagents Follow the dispenser product insert instructions to properly prime and handle the dispensers Press the dispenser caps down tightly Note Store tightly capped dispensers at 4 C when not in use 5 Check solution levels EZprep RiboWash RiboCC Reaction LCS Buffer and 1X SSC Refill if they are less than half full IMPORTANT Use reagents that have not expired 6 Empty the waste bottle it needed RNAscope VS Assay for the Discovery XT User Manual Red 25 ALD LN ADVANCED CELL DIAGNOSTICS Prepare detergent 1 Prepare 200 ml of diluted Dawn detergent by adding 4 5 ml Dawn detergent to 200 ml distilled water in a container with a cap 2 Mix well by inverting the container 4 5 times 3 Add diluted detergent to a Tissue Tek Staining Dish Note Store diluted detergent at RT Prepare mounting rea
33. r Pretreat A Ec a Pretreatment 2 Temp AB Edit Filter Low Temperature Incubation Time paa 38 Deg C iz Minutes Close W Pretreatment 3 E W Use Reaction Buffer for PT3 i Heat slides for PT 3AB Incubation Time and Temp for Pretreat B Pretreatment 3 Temp RE Low Temperature Incubation Time 38 Deg C fiz Minutes ik RNAscope VS Assay for the Discovery XT User Manual Red 17 ADVANCED CELL DIAGNOSTICS 18 Probe Hybridization Low Temperature 48 Deg C Amp 7 incubation temperature Detection 1 Temp Specify probe hybridization time and temperature PROBE 1 0761 Low Temperature L mp 3 incubation temperature Detection 3 Temp Low Temperature 155 Deg C M No Heat Amp 5 incubation time Detection 4 Plus Incubation Time 24 Minutes 55 Deg C Save As Print Clear Edit Filter Edit Filter l W Counterstain Counterstain COUNTERSTAIN 1 0741 Incubation Time 8 Minutes b W Post Counterstain Counterstain COUNTERSTAIN 2 0742 Incubation Time 4 Minutes 4 Select the appropriate assay conditions from the drop down menus according to the following table Tissue type Pretreatment 2 Pretreat A
34. r samples in every run Workflow Prepare the materials 4 Run the RNASCOPE VS Assay 10 hours 24 RNAscope VS Assay for the Discovery XT User Manual Red Prepare the materials ADVANCED CELL DIAGNOSTICS Materials can be prepared ahead of time unless otherwise stated Materials required Materials provided by Advanced Cell Diagnostics Materials provided by Ventana Medical Systems Other materials and equipment VS Target Probe e VS Positive Control Probe e VS Negative Control Probe e 10X Pretreat 2 e VS Pretreat A Red e VS Pretreat B Red e VS Amp 1 Red e VS Amp 2 Red e VS Amp 3 Red e VS Amp 4 Red e VS Amp 5 Red e VS Amp 6 Red e VS Amp 7 Red e VS Hematoxylin VS Bluing reagent Prepare the instrument DISCOVERY XT slide stainer EZPrep Buffer LCS Buffer RiboWash Buffer RiboCC Buffer used for automated cell conditioning CC2 Reaction Buffer DISCOVERY 1X SSC Option Bottle Probe dispensers mRNA Pretreatment Kit mRNA Probe Amplification Kit mRNA Red Detection Kit Generic dispensers Distilled water Dawn detergent Fume hood Xylene Tissue Tek Staining Dish 1 Tissue Tek Clearing Agent Dish xylene resistant 1 Tissue Tek Vertical 24 Slide Rack EcoMount Cover Glass 24 mm x 50 mm e If the instrument has not been used for 21 week follow the guidelines for instrument maintenance in Appendix B on page 34 e f your i
35. ral guidelines e Start with properly fixed and prepared sections Refer to Chapter 3 Prepare and Pretreat Samples on page l2 for preparation of FFPE slides For preparation of other sample types contact support acdbio com e Follow the recommended pretreatment conditions for your sample Refer to Appendix A Recommended Guidelines on page 33 for pretreatment conditions and to the technical notes available at www acdbio com technical support downloads technical doc category tech notes e Regularly maintain and clean your automated staining instrument e Always run positive and negative control probes on your sample to assess sample RNA quality and optimal permeabilization e Do nofsubstitute required materials Assay has been validated with these materials only e Follow the protocol exactly for best results e Do noflet your sections dry out during the procedure e Use good laboratory practices and follow all necessary safety procedures Refer to Appendix C Safety on page 37 for more information RNAscope VS Assay for the Discovery XT User Manual Red 11 2 Chapter 3 Prepare and Pretreat Samples Formalin tixed parattin embedded FFPE sample preparation and pretreatment are described in the following protocols IMPORTANT We highly recommend following these guidelines We cannot guarantee assay results with other preparation methods For samples treated differently from the following protocol please see the sample pre
36. salt crystallization may partially clog the valves that dispense bulk reagents possibly affecting assay performance Pertorm the following procedure fo clean the instrument and ensure optimal assay performance Check all buffer containers for any visible signs of bacterial growth Disinfect the containers with Lysol IC NEED Cat No if necessary rinse well with distilled water and refill with fresh unexpired buffers Note We recommend decontaminating the instrument and buffer containers every 3 months If you have never run an assay using these buffers perform the following procedure Flush the instrument Purge and prime the system with clean distilled water 34 Purge and prime the system with clean distilled water several times to remove debris and bacterial growth Remove all the bulk reagent containers from the instrument Connect the yellow tubing manifold usually used for quarterly decontamination to the instrument Place the open end of the tubing manifold into a bottle containing 4 L of distilled water Ensure that the tubing end touches the bottom of the bottle Note Refill the bottle with distilled water as needed 4 Click Test on the main screen and then select Function Test gt Ventana Medical Systems Discovery MT Staining Module D Addiess 0 Address iri User Logins Disabled Hosti MexES 105 Remote wizz Discovery XT Stains Serial 4 713607 Host i S200 Side Trey Po
37. scovery XT User Manual Red 27 ADVANCED CELL DIAGNOSTICS Amp 5 incubation time Detection 4 Edit Filter Plus Incubation Time 24 Minutes Close fv Counterstain Iv No Heat Counterstain Incubation Time COUNTERSTAIN 1 0741 E Minutes W Post Counterstain Counterstain Incubation Time COUNTERSTAIN 2 0742 fa Minutes 5 Select the appropriate assay conditions from the drop down menus according to the following table Tissue type Automated cell conditioning Pretreatment 2 Pretreatment 3 Brain and spinal cord 100 C mild CC2 12 MIN 12 MIN Click Save As then select a protocol number from the drop down menu and choose a protocol name for each probe Click Save Click Close to go back to the main screen Assign a probe number from the list to each probe of interest For each probe selected assign a protocol Print the labels 1 Select the Print Label icon from the bottom of the home page screen Select your preferred template or create a new template To create a new template refer fo the Ventana System User Manual tor details Select the protocol you created for the RNAscope VS Assay Click on Protocol to add and print the label Directly proceed to Loading the slides on page 29 Run the RNAscope VS Assay 28 RNAscope VS Assay
38. se 5 Check solution levels EZprep RiboWash Reaction LCS Buffer and 1X SSC Refill if they are less than half full IMPORTANT Use reagents that have not expired 6 Empty the waste bottle it needed RNAscope VS Assay for the Discovery XT User Manual Red 15 ADVANCED CELL DIAGNOSTICS Prepare detergent 1 Prepare 200 ml of diluted Dawn detergent by adding 4 5 mL Dawn detergent to 200 mL distilled water in a container with a cap 2 Mix well by inverting the container 4 5 times 3 Add diluted detergent to a Tissue Tek Staining Dish Note Store diluted detergent at RT Prepare deparaffinization reagents e In a fume hood fill two clearing agent dishes with 200 ml fresh xylene e Ina fume hood fill two staining dishes with 200 ml fresh 100 EtOH Note Ensure all containers remain covered Prepare mounting reagents e In a fume hood fill a clearing agent dish with 200 ml fresh xylene Note Ensure all containers remain covered Prepare 1X Pretreat 2 Prepare 1X Pretreat 2 while FFPE slides are baking at 60 C or the following day if you choose the optional stopping point on page 13 1X Pretreat 2 is used in manual cell conditioning CC 1 Prepare ZOO ml of fresh 1X Pretreat 2 by adding 630 ml distilled water to 1 bottle 70 ml 10X Pretreat 2 solution in the beaker 2 Mix well and cover the beaker with foil Create an instrument protocol 1 Open the NexES software and click on the Protocol bu
39. seases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S e U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cde gov biosafety e Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 2029cfr1910a_01 html e Your company s institution s Biosafety Program protocols for working with handling potentially intectious materials RNAscope VS Assay for the Discovery XT User Manual Red 37 ADVANCED CELL DIAGNOSTICS e Additional information about biohazard guidelines is available at www ede gov In the EU e Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization VHO Laboratory Biosafety Manual third edition found
40. sition no cata Reagent Tray Position mo data Three GAA T Le Pte 5 Double click on Test Purge All XT RNAscope VS Assay for the Discovery XT User Manual Red __ Ventana Medical Systems Discovery XT Staining Modula Address 0 be Download Tests Discovery XI Staining Module DISC Run Proge User Logins Disabled Host Ibs 9200 Select Tee Function to Aur Bat Code Bloo ST i Cel Conditioner AINT Zell Conditioner HANT Gowerslip DT Decontaminabe System AT Depends Reagent XT Dram Syshem sT 1 Dual Rinse S50 DST Emply Vacisum Trap XT Fhad Fath HT Hama Nogele Plate Home Reagent Tray WT gt del Dran ABIST del Drain S5C 47 Jag Nozzle Plate Cw ST Jog Reagent Trap CW WT Prime MT Purge CET WT i Puge O02 ST Pinge EZ Prep ST Puge LES ST Pinge Option T Pinge Rese Bull xT Hest NexES vIQS Side Tray Position nodata ____ RemoberviQsge O Reagent Tray Postion no data Times OA 19 2012 715 PM ADVANCED CELL DIAGNOSTICS Run E z Print l View E Protocols q Register S Eo __ Discovery XT Stains Serial 713607 __ ASD 6 You will hear a warning sound and see an error message stating that the pressue is too low This is normal Click on Sign off to turn off the warning sound 7 Repeat Step 5 four more times 8 Click Test on the main screen and then select Function Test DISC Run Progress
41. treatment optimization procedure described in Appendix A Recommended Guidelines on page 33 and technical notes available at www acdbio com technical support downloads technical doc category tech notes Prepare FFPE sections Materials required e 10 neutral buffered formalin NBF e 1X PBS e Paraffin wax e 100 ethanol EtOH e Xylene e Microtome e Water bath SuperFrost Plus slides Fix the sample 1 Immediately following dissection fix tissue in 10 NBF for 16 32 HRS at ROOM TEMPERATURE RT Fixation time will vary depending on tissue type and size A N CAUTION Handle biological specimens appropriately IMPORTANT Fixation for lt 16 HRS or gt 32 HRS will impair the performance of the RNAscope Assay Dehydrate embed and cut the sample IMPORTANT Use fresh reagents 1 Wash sample with 1X PBS Dehydrate sample using a standard ethanol series tollowed by xylene Embed sample in parattin using standard procedures Note Embedded samples may be stored at RT for years Trim parattin blocks as needed and cut embedded tissue into 5 1 ym sections using a microtome 12 RNAscope VS Assay for the Discovery XT User Manual Red ALD ADVANCED CELL DIAGNOSTICS Place paraffin ribbon in a 40 45 C water bath and mount sections on SUPERFROST PLUS SLIDES IMPORTANT Do not mount more than one section per slide Place sections in the center of the slide Air dry slides OVERNIGHT at RT OPTIONA
42. tton Click on Create Edit Protocols go to the Procedure drop down menu and select mRNA Red DXT 2 0 2 Main protocol steps appear as shown 16 RNAscope VS Assay for the Discovery XT User Manual Red ALD ADVANCED CELL DIAGNOSTICS NexES Protocol Editor Discovery XT Staining Module F List Only Registered Products Procedure mRNA Red DXT 2 0 F Filter Procedures F Wet Slide Load Baking B Deparaffinization m PreFixative T Cell Conditioning Pretreatment 2 Pretreatment 3 Specify probe hybridization time and temperature Probe Edit Filter Hybridization Low Temperature Close Amp 1 incubation temperature Detection 1 Temp Low Temperature Amp 3 incubation temperature Detection 3 Temp Low Temperature 3 After selecting the main protocol steps drop down menus become available Select the appropriate protocol steps by clicking on the associated check boxes as shown NexES Protocol Editor Discovery XT Staining Module Ed gt Pratacal a a a aaan a aN Frocedure I List Only Registered Products mRNA Red DXT 2 0 oe hi Filter Procedures Number P Wet Slide Load Baking Pre Fixative Save As Peps Sees aa l Print Jw Pretreatment 2 M Use Reaction Buffer for PT SS SSS Clear iv Heat slides for PT2 AB Incubation Time and Temp to
43. ty Storage VS Pretreat A Red Protease A 14 mL x 1 bottle 4 C VS Pretreat B Red Protease B 14 mL x 1 bottle 4 C RNAscope VS Accessory Kit Cat No 320630 w Reagent Quantity Storage VS Hematoxylin 7 mL x 1 bottle 4 C VS Bluing Reagent 7 mL x 1 bottle APC RNAscope VS Pretreat 2 Pack Cat No 320043 Reagent Quantity Storage 10X Pretreat 2 antigen retrieval solution 70 mL x 4 bottles ss Fem enone Ee IMPORTANT Do not substitute the reagent components of the RNAscope VS Reagent Kit with those of other RNAscope Reagent Kits even those having the same name 8 RNAscope VS Assay for the Discovery XT User Manual Red amp ADVANCED CELL DIAGNOSTICS Required materials from Ventana Medical Systems The RNAscope VS Assay requires specific materials and equipment available on y from Ventana Medical Systems Probe Dispensers Cat No 960 761 to 960 780 w Component Storage Probes 1 20 dispensers fill dispensers with RNAscope VS Probes Use Room temperature 20 25 C up to 20 probes at a time mRNA Pretreatment Kit Cat No 760 223 Component Storage mRNA Pretreat A dispenser fill dispenser with Pretreat A Room temperature 20 25 C mRNA Pretreat B dispenser fill dispenser with Pretreat B Room temperature 20 25 C mRNA Probe Amplification Kit Cat No 760 222 w Component Storage mRNA Red Detection Kit Cat No 760
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