- Advanced Cell Diagnostics
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1. Formalin fixed paraffin embedded FFPE sample preparation and pretreatment are described in the following protocols For other sample types and preparation methods contact support acdbio com for the latest protocols and guidelines IMPORTANT We highly recommend following these guidelines We cannot guarantee assay results with other preparation methods For suboptimally treated samples you may need to optimize pretreatment conditions Refer to Appendix A Tissue Pretreatment Recommendations on page 19 Prepare FFPE sections Materials required e 10 neutral buffered formalin NBF e LN PBS e Paraffin wax e 100 ethanol ACS grade or equivalent e Xylene e Microtome e Water bath e SuperFrost Plus slides Fix the sample 1 Remove sample and CUT 3 4 mm pieces prior to fixing ra CAUTION Handle biological specimens appropriately 2 Fix sample in FRESH 10 NBF for 16 32 HOURS at ROOM TEMPERATURE Fixation time will vary depending on tissue type IMPORTANT Under fixation will result in significant signal loss when performing the RNAscope Assay Sample Preparation and Pretreatment Guide for FFPE Tissue 11 AED Dehydrate embed and cut the sample IMPORTANT Use fresh reagents WASH sample with 1X PBS DEHYDRATE sample using a standard ETHANOL series followed by XYLENE EMBED sample in PARAFFIN Note Embedded samples may be stored at room temperature for years Trim paraff2. Placenta Normal Standard Skin TMA Normal Standard Breast TMA Normal Standard Melanoma TMA Normal Standard Nevus TMA Benign Standard Stomach TMA Normal Standard Stomach TMA Tumor Standard Cell Pellets fixed with 10 NBF Mild HeLa cells fixed with 10 L Standard Formaldehyde PBS ACD Control Tissue Microarray 20 Sample Preparation and Pretreatment Guide for FFPE Tissue Determine reagent volume Appendix B Reagent Volume Guidelines Before starting your experiment measure the inner edge of the hydrophobic barrier to determine the recommended number of drops needed per slide see table below Size of Recommended Recommended Relative template size hyrophobic number of drops volume per slide barrier in per slide pL 0 75 x 0 75 t 4 120 0 75 x 1 0 5 150 0 75 x 1 25 6 180 Hydrophobic barrier measured at inner edge References in this user manual are for the 0 75 x 0 75 hydrophobic barrier size t Recommended hydrophobic barrier size is 0 75 x 0 75 With this barrier size each probe is sufficient for staining 20 sections Larger tissue sections will result in fewer tests Sample Preparation and Pretreatment Guide for FFPE Tissue 21 AED ADVANCED CELL DIAGNOSTICS INC 22 Sample Preparation and Pretreatment Guide for FFPE Tissue e Appendix C Safety Che
3. for FFPE Tissue Chapter 2 Before You Begin Prior to running the RNAscope Assay on your samples for the first time we recommend that you e View the video demonstrations available at www acdbio com 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 3T3 using the Positive and Negative Control Probes Important procedural guidelines e Start with properly fixed and prepared sections Refer to Chapter 3 Prepare and Pretreat Samples on page 11 for preparation of FFPE slides For preparation of other sample types contact support acdbio com e Follow the recommended pretreatment guidelines for your sample Refer to Appendix A Tissue Pretreatment Recommendations on page 19 for pretreatment conditions e Always run positive and negative control probes on your sample to assess sample RNA quality and optimal permeabilization e Do not substitute required materials Assay has been validated with these materials only e Follow the protocol exactly for best results e Use good laboratory practices and follow all necessary safety procedures Refer to Appendix C Safety on page 23 for more information Sample Preparation and Pretreatment Guide for FFPE Tissue 9 AED ADVANCED CELL DIAGNOSTICS INC 10 Sample Preparation and Pretreatment Guide for FFPE Tissue AED Chapter 3 Prepare and Pretreat Samples
4. require less time For these tissue types vary the PRETREAT 2 TIME to 8 minutes and PRETREAT 3 TIME to 15 minutes If you have a tissue type not listed contact support at support acdbio com Tissue specific pretreatment conditions If your sample fixation is successful in fresh 1076 NBF Step 2 above then refer to the following table for tissue specific pretreatment conditions For information about species or tissue type not listed here contact support at support acdbio com Species Tissue type Pathology Pretreatment Condition Mouse Rat Intestine Normal Standard Intestine Tumor Standard Embryo Normal Standard Brain Normal Standard Spleen Normal Mild Sample Preparation and Pretreatment Guide for FFPE Tissue 19 AED Species Tissue type Pathology Pretreatment Condition Eye Retina Normal Standard Liver Normal Extended Kidney Normal Standard Human Breast Tumor Standard Colon T umor Standard Colon Normal Standard Lung Tumor Standard Lung Normal Standard Prostate Tumor Standard Prostate Normal Standard Lymph node Tumor Mild Lymph node Normal Mild Tonsil Normal Mild Pancreas Normal Standard Cervical Cancer Standard Cervical Normal Standard Cervical dysplasia Abnormal Standard Brain Tumor Standard Brain Normal Standard Head Cancer Standard Neck Cancer Standard Liver Cancer Standard Kidney Normal Standard Skin Normal Standard Melanoma Tumor Standard Nevus Benign Standard
5. section OPTIONAL STOPPING POINT 4 Dry slides at room temperature overnight for use the following day must use within 24 hours or proceed directly to the next section Sample Preparation and Pretreatment Guide for FFPE Tissue Apply Pretreat 3 1 Place the dried slides on the HybEZ Slide Rack and add 5 DROPS of PRETREAT 3 to entirely cover each section 2 Remove the HybEZ Humidity Control Tray from the HybEZ Oven and place the HybEZ Slide Rack in the tray Close the lid and insert tray back into the oven INCUBATE at 40 C for the amount of time specified by the table in Appendix A Tissue Pretreatment Recommendation on page 19 Note PREPARE RNASCOPE 2 0 assay materials during this step 3 Remove the HybEZ Humidity Control Tray from the oven Remove the HybEZ Slide Rack from the tray 4 Take each slide one at a time from the HybEZ Slide Rack and tap and or flick to remove the excess liquid Immediately place each slide in a Tissue Tek Slide Rack submerged in a Tissue Tek Staining Dish filled with DISTILLED WATER 5 WASH slides with distilled water by moving the rack up and down 3 5 TIMES and REPEAT with FRESH DISTILLED WATER Proceed to the RNAscope Assay While slides are incubating proceed immediately to an RNAscope Assay Ensure you have the appropriate user manual User manuals are available at www acdbio com support technical doc For any questions please contact support acdbio c
6. LLED WATER 3 Insert covered tray into oven and close the oven door WARM the TRAY for 30 MINUTES at 40 C before use Prepare 1X Pretreat 2 IMPORTANT Do not boil 1X Pretreat 2 more than 30 minutes before use 1 Prepare 700 mL of FRESH 1X PRETREAT 2 by adding 630 mL DISTILLED WATER to 1 bottle 70 mL 10X Pretreat 2 solution in a 1 L beaker Mix well 2 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 MINUTES 3 Once 1X Pretreat 2 reaches boiling turn the hot plate knob to 100 104 C to maintain uniform boiling Apply Pretreat 1 1 Lay deparaffinized slides on the bench and add 5 8 DROPS of PRETREAT 1 to cover the entire section IMPORTANT Ensure the slides are placed on a leveled surface 2 Incubate slides for 10 MINUTES at ROOM TEMPERATURE Sample Preparation and Pretreatment Guide for FFPE Tissue 15 Apply Pretreat 2 Remove Pretreat 1 solution from one slide at a time by tapping the slide on absorbent paper and or flicking Immediately insert the slide into a Tissue Tek Slide Rack submerged in a Tissue Tek Staining Dish filled with DISTILLED WATER WASH slides in the DISTILLED WATER by moving the rack up and down 3 5 TIMES and REPEAT with FRESH DISTILLED WATER Ensure that 1X PRETREAT 2 solution is at mild boiling With a pair of forceps very slowly submerge the slide rack containing the slides into the boiling 1X P
7. Pretreat 2 Turn on HybEZ OVEN i Place a Humidifying Paper in the Humidity Control Tray i Insert covered tray into oven and close the oven door WARM the TRAY for 30 MINUTES at 40 C before use Prepare 1X Pretreat 2 10 20 minutes Apply Pretreat 1 10 minutes i Apply Pretreat 2 10 30 minutes i Create Hydrophobic barrier Optional Overnight Stopping Point 4 Apply Pretreat 3 30 60 minutes Proceed IMMEDIATELY to the RNAscope 2 0 Assay Immediately proceed to the RNAscope Assay Chapter of the appropriate Part 2 User Manual RNAscope 2 0 HD Detection Kit BROWN Catalog No 320497 RNAscope 2 0 HD Detection Kit RED Catalog No 320487 and RNAscope 2 Plex Detection Kit Chromogenic Catalog No 320494 14 Sample Preparation and Pretreatment Guide for FFPE Tissue Materials required Materials provided by Advanced Cell Other materials and equipment Diagnostics e Pretreat 1 e Prepared slides e Pretreat 2 e Distilled water e Pretreat 3 e Glass beaker 1 or 2 L e HybEZ Humidifying System e Paper towel or absorbent paper e Hot plate isotemp brand e Aluminum foil e Thermometer e Forceps large e Thermometer e Tissue Tek Slide Rack e Tissue Tek Staining Dish Equilibrate equipment 1 Turn on HybEZ OVEN and set temperature to 40 C 2 Place a Humidifying Paper in the Humidity Control Tray and wet completely with DISTI
8. RETREAT 2 solution Cover the beaker with foil and BOIL the slides for the amount of time specified by the table in Appendix A Tissue Pretreatment Recommendations on page 19 After pretreatment time is over 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 in the distilled water by moving the rack up and down 3 5 TIMES and REPEAT with FRESH DISTILLED WATER WASH slides in FRESH 100 ETOH by moving the rack up and down 3 5 TIMES Air dry the slides Create a hydrophobic barrier 16 1 Use the following template to DRAW a barrier 2 4 TIMES around each section with the IMMEDGE HYDROPHOBIC BARRIER PEN See example below size of this hydrophobic barrier is 0 75 x 0 75 Note Refer to Appendix B Reagent Volume Guidelines on page 21 to determine the recommended number of drops needed per slide IMPORTANT Do not let the barrier touch the section Tissue Slide Label Note We do not recommend drawing a smaller barrier and using less than the recommended volume amounts even for smaller sections Larger barriers will result in fewer tests per kit Let the barrier DRY completely 2 MINUTES or OVERNIGHT at Room Temperature Note If you need to reapply the hydrophobic barrier during the following procedures dry the appropriate area of the slide with a kimwipe Do not touch the tissue
9. USER MANUAL 9 WI ADVANCED CELL DIAGNOSTICS INC RNAscope Sample Preparation and Pretreatment Guide for FFPE Tissue PART 1 Catalog Number 32051 1 Compatible with the following User Manuals RNAscope 2 0 HD Detection Kit BROWN Catalog No 320497 RNAscope 2 0 HD Detection Kit RED Catalog No 320487 and RNAscope 2 Plex Detection Kit Chromogenic Catalog No 320494 For Molecular Biology Applications not intended for diagnosis Rev Date 20130628 For Molecular Biology Applications not intended for diagnosis Trademarks RNAscope and HybEZ are trademarks of Advanced Cell Diagnostics Inc All other trademarks belong to their respective owners Citing RNAscope 2 0 in Publications When describing a procedure for publication using this product please refer to it as the RNAscope 2 0 Assay and cite Wang F Flanagan J Su N Wang L C Bui S Nielson A Wu X Vo H T Ma X J and Luo Y RNAscope A Novel n Situ RNA Analysis Platform for Formalin Fixed Paraffin 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 from the
10. aster Tech LWSRA24 Tek Tissue Tek Vertical 24 Slide Rack Scientific MLS American Master Tech Tek Tissue Tek Staining Dish 6 required Scientific MLS LWT4457EA Tissue Tek9 Clearing Agent Dish xylene resistant American Master Tech 3 required Scientific IMLS SOCIO eM Cytoseal XYL xylene based mounting medium Richard Allen Scientific MLS 9312 4 Cover Glass 24 x 50 mm Fisher Scientific MLS 12 545 F Sample Preparation and Pretreatment Guide for FFPE Tissue AED kd Description Supplier Cat no Ammonium hydroxide 28 30 Sigma Aldrich MLS 320145 500mL Carboy gt 3L MLS Water bath or incubator capable of holding MLS temperature at 40 1 C Pipettors and tips 1 1000 uL MLS Distilled water MLS ubes various sizes MLS Fume hood MLS Graduated cylinder MLS Parafilm MLS Paper towel or absorbent paper MLS Glass beaker 1 or 2 L MLS Hot plate Fisher Scientific MLS 11 300 49SHP Aluminum foil MLS Forceps large MLS Thermometer MLS 20 bleach MLS es Microscope and accessories MLS ES 10 neutral buffered formalin NBF MLS E Paraffin wax MLS Microtome MLS p MLS Drying oven capable of holding temperature at 60 1 C Major Laboratory Supplier in North America For other regions please check Catalog Numbers with your local lab supplier Sample Preparation and Pretreatment Guide
11. d on the ACD website at www acdbio com tos terms and conditions of sale If you have any questions please contact Advanced Cell Diagnostics at www acdbio com support Sample Preparation and Pretreatment Guide for FFPE Tissue 25 Headquarters 3960 Point Eden Way Hayward CA 94545 Phone 1 510 576 8800 Toll Free 1 877 576 3636 9 For support email support acdbio com Wo ADVANCED CELL DIAGNOSTICS INC www acdbio com
12. e 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 Sample Preparation and Pretreatment Guide for FFPE Tissue 23 24 In the U S In the EU materials Read and follow the applicable guidelines and or regulatory requirements in the following U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 2029cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety who cds c
13. eeseeenee enne nnne 15 Prepare 1X EC 15 Apply Pretreat EE 15 Apply dE 16 Create a hydrophobic baier 16 Apply Pretreat 3 ise idees peritis e edle open 17 Proceed to the RNAscope Assay c cccceecseseeseeeseeeeeeeeeeeecaesaeeeeeeaeeaeens 17 Troubleshoo lingsen 17 Appendix A Tissue Pretreatment Recommendation 19 Tissue pretreatment recommendation eene 19 Tissue specific pretreatment conditions eee eee ete eee seen tenes 19 Appendix B Reagent Volume Guidelines 21 Determine reagent volume nennen 21 Appendix C Safety rnit citra nante nk S RR Ree pa cEE EE RRKES 23 Sample Preparation and Pretreatment Guide for FFPE Tissue 3 Chemical safety EE 23 Biological hazard E 3 tt tret eese ar ru eonun 23 Documentation and support eeeeeeeeeeeeeeeenennnnn 25 Obtaining MSD SS ette oa eite eec retis tea RE Ea et e e Decades 25 Obtaining BET Te 25 Contact informatioh 2 cect cette ntere t iiaia 25 Limited product warranty esee 25 Sample Preparation and Pretreatment Guide for FFPE Tissue Chapter 1 Product Information Before using this product read and understand the information in Appendix C Safety on page 23 in this document IMPORTANT We recommend reading the entire user manual before beginning any protocols About this guide This user manual provides guidelines and protoco
14. in blocks as needed and CUT embedded tissue into 5 1 um sections using a microtome Place paraffin ribbon in water bath and MOUNT sections on SUPERFROST PLUS SLIDES IMPORTANT Place sections in the center of the slide AIR DRY slides OVERNIGHT at ROOM TEMPERATURE Do NOT bake slides unless they will be used within 1 week OPTIONAL STOPPING POINT 1 Use sectioned tissue within 3 months Store sections with dessicants at room temperature Prepare FFPE slides for the RNAscope Assay Workflow 12 Bake slides 1 hour i Optional 1 week Stopping Point 2 i Deparaffinize FFPE tissue 20 minutes Optional Overnight Stopping Point 3 Sample Preparation and Pretreatment Guide for FFPE Tissue Materials required Drying oven Prepared FFPE slides Tissue Tek Vertical 24 Slide Rack Distilled water Fume hood Xylene 100 ethanol Tissue Tek Clearing Agent Dish 2 Tissue Tek Staining Dish 2 ImmEdge Hydrophobic Barrier Pen Bake slides Bake slides in a dry oven for 1 HOUR at 60 C OPTIONAL STOPPING POINT 2 Use sectioned tissue within 1 week Store sections with dessicants at room temperature If you continue prepare the materials for the following protocols while the slides are baking Deparaffinize FFPE sections on page 13 and PRETREAT SAMPLES on page 15 Deparaffinize FFPE sections Reagents may be prepared ahead of time Ensure all containers
15. is system only The HybEZ Hybridization System 110 VAC Cat no 310010 220 VAC Cat no 310013 is designed for the hybridization and incubation steps in the RNAscope Assays Incubation steps in the RNAscope Assay require humid conditions to prevent sections from drying out For instructions on how to use the HybEZ Hybridization System refer to the HybEZ Hybridization System User Manual available at www acdbio com support technical doc and view the training video at www acdbio com support online training videos The system contains the following components Component Quantity Cat no HybEZ Oven 110 or 220 VAC 1 oven 310010 or 310013 HybEZ Humidity Control Tray with lid 1 tray 310012 HybEZ Slide Rack 20 slide capacity 1 rack 310014 HybEZ Humidifying Paper 2 sheets HybEZ Humidifying Paper Pack 15 sheets 310015 User supplied materials IMPORTANT Do not substitute other materials for the ImmEdge Hydrophobic Barrier Pen and the SuperFrost Plus Slides listed in the following table Description Supplier Cat no ImmEdge Hydrophobic Barrier Pen required Vector Laboratory H 4000 SuperFrost Plus Slides required Fisher Scientific 12 550 15 100 ethanol American Master Tech ALREAGAL Scientific MLS Gill s Hematoxylin American Master Tech HXGHE1LT Scientific MLS Xylene Fisher Scientific MLS X3P 1GAL e American M
16. ls for the proper preparation and pretreatment of formalin fixed paraffin embedded FFPE tissues mounted on slides The slides can then be assayed using an RNAscope Detection Kit RNAscope Reagent Kits come with a separate RNAscope Assay User Manual Do not perform an RNAscope Assay without the correct user manual Refer to the manuals available at www acdbio com support technical doc for more details For any questions please contact support acdbio com Product description Background Sample types The RNAscope Assays use a novel and proprietary method of in situ hybridization ISH to visualize single RNA molecules per cell in samples mounted on slides The assays are based on ACD s patented signal amplification and background suppression technology Proprietary RNA specific probes are hybridized to target RNA and are then bound to a cascade of signal amplification molecules culminating in signal detection Figure 1 Single plex 2 plex multiplex and automated assays are all available The RNAscope Assay procedure is illustrated in Figure 1 on page 6 and can be completed in 6 10 hours depending on the assay or conveniently divided over two days Most RNAscope Assay reagents are available in convenient Ready To Use RTU dropper bottles and provide a simple nearly pipette free workflow Results are observable using standard bright field or fluorescent microscopy In order to perform the RNAscope Assays you must start with
17. mical safety A 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 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 Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Biological hazard safety AN WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicabl
18. om Note Slides can stay in distilled water for up to 15 minutes IMPORTANT Slides should not stay in distilled water for longer than 15 minutes Proceed to the RNAscope Assay using the appropriate user manual Troubleshooting For troubleshooting information refer to the Troubleshooting User Manual Cat no 320519 available at www acdbio com support technical doc For any questions please contact support acdbio com Sample Preparation and Pretreatment Guide for FFPE Tissue 17 AED ADVANCED CELL DIAGNOSTICS INC 18 Sample Preparation and Pretreatment Guide for FFPE Tissue Appendix A Tissue Pretreatment Recommendation Follow the recommended pretreatment conditions based on your tissue type for e Any new or previously untested FFPE tissue types e Suboptimally prepared samples Tissue pretreatment recommendation 1 Stain representative samples using the positive and negative control probes 2 Fixsample in FRESH 1076 NBF for 16 32 HOURS at ROOM TEMPERATURE Note Perform tissue fixation step using the recommended amount of time Over or under fixation will result in significant signal loss when performing the RNAscope Assay 3 Depending on your tissue type see section below vary the PRETREAT 2 and or PRETREAT 3 TIME Reagent Mild Standard Extended Pretreat 2 15 min 15 min 30 min Pretreat 3 15 min 30 min 30 min Note Sample types such as certain Xenografts and Cell Pellets
19. product to find a gene specific probe from a searchable catalog of gt 27 000 predesigned Target Probes or order a custom probe Visit www acdbio com products target probes controls housekeeping to find appropriate Control Probes RNAscope Reagent Kit Each RNAscope Detection Kit provides enough reagents to stain 20 tissue sections approximately 20 mm x 20 mm large Larger tissue sections will result in fewer tests Each kit contains three sub kits including a Pretreatment Kit Cat no 310020 Pretreatment Kit instructions are provided in this guide For information on the other sub kits and directions for use refer to an RNAscope Detection Kit User Manual The reagents have a shelf life of six months from the shipment date when stored as indicated in the following table Pretreatment Kit Cat no 310020 Reagent Quantity Storage Pretreat 1 Ready To Use RTU endogenous blocker 4 mL x 2 bottles 4 C 10X Pretreat 2 70 mL x 4 bottles Room temperature 20 25 C Pretreat 3 RTUt protease 4 5 mL x 1 bottle 4 C Comes in a separate box t Dilute Pretreat 3 for some samples See Appendix A Tissue Pretreatment Guidelines on page 19 Sample Preparation and Pretreatment Guide for FFPE Tissue Required materials and equipment The following materials and equipment are needed to perform the RNAscope Assay HybEZ Hybridization System IMPORTANT The RNAscope Assay has been validated using th
20. properly prepared and pretreated samples Multiple sample types are now compatible with RNAscope Assays and include formalin fixed paraffin embedded FFPE tissues fresh frozen tissues fixed frozen tissues tissue microarray TMA and cultured cells Visit www acdbio com and contact support acdbio com for more information Sample Preparation and Pretreatment Guide for FFPE Tissue 5 AED ADVANCED CELL DIAGNOSTICS INC Figure 1 Procedure overview ILS r AMP abel Probe ZZ Target RNA Specific Oligo Probes z Zz z a ilt z Ex Ua 2 Hybridize to target RNA Hybridize gene specific probe pairs to the target mRNA 1 Tissue section 3 Amplify signal Probes are hybridized to a cascade of signal amplification molecules culminating in binding of HRP or AP labeled probes The 2 0 Assay enhances signal further with additional amplification steps Add DAB or Fast Red substrate to detect target RNA Visualize target RNA using a standard bright field microscope Start with properly prepared sections and pretreat to allow access to target RNA RNAscope Assay reagents RNAscope Assays require the RNAscope Probes and an RNAscope Detection Kit If you are performing an automated assay using the Ventana DISCOVERY XT or ULTRA Systems you must use RNAscope VS Probes and the RNAscope VS Reagent Kit Probes and Reagent Kits are available separately Visitwww acdbio com products target probes search
21. remain covered 1 Ina fume hood fill TWO Clearing Agent DISHES with 200 mL FRESH XYLENE In a fume hood fill TWO Staining DISHES with 200 mL FRESH 100 ETHANOL 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 MINUTES at ROOM TEMPERATURE Agitate the slides by occasionally lifting the slide rack up and down in the Clearing Agent Dish Remove the slide rack from the first xylene containing dish and immediately place in the second xylene containing Clearing Agent Dish in the fume hood REPEAT STEP 2 Remove the slide rack from the second xylene containing dish and immediately place in the Staining Dish containing 100 ethanol Incubate the slides in 100 ETHANOL for 1 MINUTE at ROOM TEMPERATURE with agitation REPEAT STEP 6 with FRESH 100 ETHANOL Remove the slides from the rack and place on absorbent paper with the section face up AIR DRY for 5 MINUTES at ROOM TEMPERATURE OPTIONAL STOPPING POINT 3 Air dry overnight at room temperature must use within 24 hours or proceed directly to the next step Sample Preparation and Pretreatment Guide for FFPE Tissue 13 Pretreat samples IMPORTANT Before you begin make sure you know the pretreatment conditions specific to your sample type Refer to Appendix A Tissue Pretreatment Recommendations on page 19 Workflow Equilibrate equipment and prepare 1X
22. sr lyo 2004 11 en Information about the Registration Evaluation Authorisation and Restriction of Chemicals REACH can be found at eur lex europa eu LexUriServ LexUriServ do uri O L 2010 133 0001 0043 EN PDF Sample Preparation and Pretreatment Guide for FFPE Tissue ACD Documentation and support Obtaining MSDSs Material Safety Data Sheets MSDSs are available at www acdbio com support technical doc category msds 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 support 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 affiliate s warrant their products as set forth in the ACD General Terms and Conditions of Sale foun
23. use of this information Copyright 2013 Advanced Cell Diagnostics Inc All rights reserved AED Contents Chapter 1 Product Information eese 5 ADOUU TMS GUI E 5 Product Ee IEN 5 Background EP 5 Sample rc EC dente 5 RNAscope Assay reagents ss sesseeesirenesrstnttetnttnt nt tntart nt atnnnr anenee enen 6 RNAscope Reagent kt 6 Required materials and equipment sseseeeeeen 7 HybEZ Hybridization System sss 7 User supplied materials seen nnne 7 Chapter 2 Before You Begin nere 9 Important procedural guidelines aasssseesssreessrresssrrnsrsnnenrnnnenrnnnnsnnnnesnnnnnnnenne 9 Chapter 3 Prepare and Pretreat Samples 11 Prepare PFPE sections ronis Eu rne ese ie ers D e rua PE e edes 11 Materials required eeseseesesseseeseeeseeee seen 11 aos Xe 11 Dehydrate embed and cut the sample sss 12 Prepare FFPE slides for the RNAscope Assay ssssssseee 12 lale E 12 Materials required Tm 13 el E 13 Deparaffinize FFPE sections esee 13 Pretreat samples sxc ies dete eee Lb Code Ehe Urn eI eos Eee ED eaae ea fain 14 WOPKOW EL 14 Materials required EE 15 Equilibrate equipment ceeeessceeeessceeee
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