- Advanced Cell Diagnostics
Contents
1. Use sectioned tissue within 3 months Prepare the slides for the RNAscope Assay Workflow Fix the sections 15 MIN i Dehydrate the sections 20 MIN i Optional Stopping Point 2 Create a hydrophobic barrier 10 MIN i Apply Pretreat 4 30 MIN i Proceed IMMEDIATELY to the RNAscope Assay Immediately proceed to the RNAscope Fluorescent Multiplex Detection Kit Part 2 User Manual Catalog No 320293 12 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue Materials required e 1X PBS e 1096 NBF or 496 PFA e 10096 ethanol EtOH e Tissue Tek Vertical 24 Slide Rack e Tissue Tek Staining Dishes 5 required e ImmEdge Hydrophobic Barrier Pen Fix the sections Chill 200 mL 10 NBF fresh 10 NBF or 4 PFA in 1X PBS to 4 C in a Tissue Tek Staining Dish Remove slides from 80 C and place in a Tissue Tek Slide Rack Immediately immerse slides in the pre chilled fixative Fix for 15 MIN at 4 C Note Formalin that has been stored for more than 6 months exposed to air for more than a week or used repeatedly may result in suboptimal tissue fixation Dehydrate the sections Reagents may be prepared ahead of time Ensure all containers remain covered IE UL o 2 JT Prepare 200 mL 50 EtOH 200 mL 70 EtOH and 600 mL 100 EtOH in Tissue Tek Staining Dishes Place the slides in 50 EtOH for 5 MIN at ROOM TEMPERATURE RT Place the slid2. 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 csr 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 OJ L 2010 133 0001 0043 EN PDF Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue AED ANCED CELL DIAGNOSTICS IN 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 or
3. NT Do not apply Pretreat 1 or Pretreat 2 to fresh frozen sections when using the RNAscope Fluorescent Multiplex Kit Other sample types may need these treatments Materials required Materials provided by the RNAscope Other materials and equipment Reagent Kit e Pretreat 3 amp 4 e Prepared slides e Distilled water e Paper towel or absorbent paper e HybEZ Humidifying System e Tissue Tek Slide Rack e Tissue Tek Staining Dish Prepare the equipment e Turn on HybEZ Oven and set temperature to 40 C e Place a Humidifying Paper in the Humidity Control Tray and wet completely with distilled water Apply Pretreat 4 IMPORTANT View the wash step video at www acdbio com support online training videos wash slides before proceeding 1 Place the dried slides on the HybEZ Slide Rack and add 5 drops of Pretreat 4 to entirely cover each section 2 Incubate for 30 MIN at RT 14 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue AED ANCED CELL DIAGNOSTICS INE 3 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 1X PBS 4 Wash slides with 1X PBS by moving the rack up and down 3 5 times and repeat with 1X PBS Proceed to the RNAscope Assay While slides are incubating proceed immediately to the appropriate RNAscope A
4. USER MANUAL AG ye PED ADVANCED CELL DIAGNOSTICS INC RNAscope Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue For Manual RNAscope Assay PART 1 Catalog Number 320513 Compatible with the RNAscope Fluorescent Multiplex Detection Kit Part 2 Catalog No 320293 For Molecular Biology Applications not intended for diagnosis Rev Date 20131105 For Molecular Biology Applications 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 In 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 use of this information Copyright 2013 Advanced Cell Diagnostics Inc All rights reserved Chapter 1 Product Infor
5. ble at www acdbio com product literature html and view the training video at www acdbio com demo video html The system contains the following components Component Quantity Cat No EEL int 310012 ALL pun 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 EtOH ALREAGAL Scientific MLS Cryo embedding medium e g OCT NC9806257 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue a 3 Description Supplier Cat No Cryomold Fisher Scientific MLS NC9511236 10 neutral buffered formalin NBF or Fisher Scientific MLS 23 245 684 4 paraformaldehyde PFA Cryostat capable of cooling down to at least MLS 20 C Slide boxes MLS Aluminum foil or zip lock bags MLS l American Master Tech LWSRA24 Tissue Tek Vertical 24 Slide Rack Scientific MLS l Bana l American Master Tech Tissue Tek Staining Dish 6 required ecientific MLS LWT4457EA Water bath or incubator capable of holding MLS temperature at 40 1 C Distilled water MLS Paper towel or absorbent paper MLS Major Laboratory Supplier in North America For other regions please check Catalog Nu
6. cal samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases 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 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue 19 ASD 20 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
7. ders 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 found 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 Fresh Frozen Tissue Headquarters 3960 Point Eden Way Hayward CA 94545 Phone 1 510 576 8800 Toll Free 1 877 576 3636 E For support email support acdbio com Wo ADVANCED CELL DIAGNOSTICS INC www acdbio com
8. e Forceps e Cryo embedding medium OCT e Dry ice liquid nitrogen or isopentane e Cryostat e Slide boxes e SuperFrost Plus slides e Aluminum foil or zip lock bags Prepare the block 1 Remove tissue and cut to fit into cryomolds AN CAUTION Handle biological specimens appropriately 2 Freeze the specimen on dry ice or in liquid nitrogen within 5 MIN of tissue harvest 3 Embed frozen tissue in cryo embedding medium OCT a Add two drops of OCT into a cryomold b Place the frozen tissue on the OCT in the correct orientation for cutting C Add more OCT to fill the cryomold Do not allow any air bubbles to form d Hold the block with forceps on the surface of liquid nitrogen or isopentane cooled by dry ice or liquid nitrogen or place the cryomold on dry ice 4 Store the frozen block in an air tight container at 80 C prior to sectioning Note Embedded tissue may be stored for at least 3 MONTHS Section the block 1 Equilibrate block to 20 C in a cryostat 1 HR Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue 11 AD ADVANCED CELL DIAGNOSTICS IN 2 Cut 10 20 um sections and mount onto SuperFrost Plus slides 3 Keep the sections at 20 C to dry 4 Store the sections in slide boxes wrapped air tight with aluminum foil or zip lock bags at 80 C until use Note Sections may be stored for at least 3 MONTHS IMPORTANT Do not fix the slides prior to this step OPTIONAL STOPPING POINT 1
9. en ennnennnnnenn eens Biological hazard safety sssseuuuse Documentation and support Obtaining MSDSS ssssvevtiencis iste TERR IXEER TA FhYX ERE R Cui UE EDS Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue AED Contents AD ADVANCED CELL DIAGNOSTICS INC Obtaining support eeeeesssssssesesseeeneeeennnnnen nnne nennen nnne nar nnn nnns 21 Contact information sssssssesssessssesseeene nennen nnne nnnm nain nnn nnns 21 Limitea prod ct Waranty ase erscies cca thetaucnecniaeni iniia a aai 21 4 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue ASD Chapter 1 Product Information Before using this product read and understand the information in Appendix B Safety on page 19 in this document IMPORTANT We recommend reading the entire user manual before beginning any protocols About this guide This user guide provides guidelines and protocols for the proper preparation and pretreatment of fresh frozen tissues mounted on slides The slides can then be assayed using an RNAscope Reagent Kit RNAscope Reagent Kits come with a separate RNAscope Assay User Manual Do not perform an RNAscope Assay without the correct user manual Product description Background The RNAscope Assays use a novel and proprietary method of in situ hybridization ISH to visualize single RNA molecules per cell in sample
10. es in 70 EtOH for 5 MIN at RT Place the slides in 100 EtOH for 5 MIN at RT Repeat step 6 with fresh 100 EtOH OPTIONAL STOPPING POINT 2 Slides may be stored in 100 EtOH at 20 C for up to 1 WEEK Prolonged storage may degrade sample RNA Create a hydrophobic barrier 1 Take slides out of 100 EtOH and place on absorbent paper with the section face up Air dry for 5 MIN at RT 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 A Reagent Volume Guidelines on page 17 to determine the recommended number of drops needed per slide IMPORTANT Do not let the barrier touch the section Immedge hydrophobic barrier pen is highly recommended Alternative type of pen may result in suboptimal results Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue 13 Hydrophobic Barrier 0 75 x0 75 Tissue Slide L abel 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 3 Let the barrier dry completely 1 MIN 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 section Pretreat samples IMPORTA
11. et RNA 3 Amplify signal Start with properly prepared Hybridize multiple sets of Use up to four signal amplification systems to Visualize target RNA tissue sections and pretreat gene specific probe pairs to detect multiple target RNAs Probes are using a standard to allow access to target target mRNAs hybridized to a cascade of signal amplification fluorescent RNA molecules culminating in binding of dye labeled microscope probes visible in different fluorescent chanels RNAscope Assay reagents RNAscope Assays require the RNAscope Probes and an RNAscope Reagent Kit If you are performing an automated assay using the Ventana DISCOVERY XT or ULTRA Systems you must use RN Ascope VS Probes and the RNAscope VS Reagent Kit Probes and Reagent Kits are available separately Visit www acdbio com products target probes search 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 Reagent Kit provides enough reagents to stain 20 sections each with an area of approximately 20 mm x 20 mm 0 75 x 0 75 Larger tissue sections will result in fewer tests Each kit contains three sub kits including a Pretreatment Kit which come with up to three reagents Fresh frozen tissue Cat No 320842 requires only Pretreat 4 Pretrea
12. mation About this guide naan enn eennnneenn eenn Product description nanne eenn eennnn eener ennen BACKOFOUNO M Sample types eseeeeeneeeennenmn RNAscope Assay reagents eee RNAscope Reagent Kit neen Required materials and equipment HybEZ Hybridization System User supplied materials Chapter 2 Before You Begin Important procedural guidelines Chapter 3 Prepare and Pretreat Samples Prepare fresh frozen sections Materials required nanne senen enn enen Prepare the block annae eennn enne eenen Section the block aaan eennnnenn Prepare the slides for the RNAscope Assay WY ORO T Materials required an aen enn ennn enn enen Fix the sections aaan an nen ene eenen e ennen vennen Dehydrate the sections annees enene enen Create a hydrophobic barrier Pretreat samples nanne en nennen vena enn venen Materials required naan ennen enn enen Prepare the equipment annen enen Apply Pretreat 4 nanne vennen Proceed to the RNAscope Assay nennen Troubleshooting nnen enn eeens venn Appendix A Reagent Volume Guidelines Determine reagent volume annen eneen Appendix B Safety Chemical safety nanos enn
13. mbers with your local lab supplier 8 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue ASD 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 Important procedural guidelines e Start with properly fixed and prepared sections 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 B Safety on page 19 for more information Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue AD ADVANCED CELL DIAGNOSTICS INC 10 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue AED Chapter 3 Prepare and Pretreat Samples Fresh frozen 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 Prepare fresh frozen sections Materials required e Scalpel
14. or Fresh Frozen Tissue 17 AD ADVANCED CELL DIAGNOSTICS INC 18 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue ASD Appendix B Safety Chemical safety AN 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 Biologi
15. s 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 Sample types In order to perform the RNAscope Assays you must start with properly prepared and pretreated samples Multiple sample types are now compatible with RNAscope Assays and include formalin fixed paraffin embedded FFPE tissue fresh frozen FF tissue fixed frozen tissue tissue microarray TMA and cell samples Visit www acdbio com and contact support acdbio com for more information Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue AD ADVANCED CELL DIAGNOSTICS INC Figure 1 Procedure overview Z Z Target RNA Specific Oligo Probes a PreAMP AMP Label Probe Z Z2 pe zz z z z ei YY MED FF mRNA1 mRNA2 1 Tissue section 2 Hybridize to targ
16. ssay protocol 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 com Note Slides can stay in 1X PBS for up to 15 MIN IMPORTANT Slides should not stay in 1X PBS for longer than 15 MIN Proceed to the RNAscope Assay using the appropriate user manual Troubleshooting For troubleshooting information please contact technical support at support acdbio com Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue 15 AD ADVANCED CELL DIAGNOSTICS INC 16 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue AED LL DIAGNOSTI A Appendix A Reagent Volume Guidelines Determine reagent volume 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 uL B E 180 0 75 x 1 25 6 um 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 f
17. tment Kit instructions are provided in this guide For information on the other sub kits and directions for use refer to an RNAscope Assay 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 320842 Pretreat 3 1X protease 4 5 mL x 1 bottle Pretreat 4 2X protease 4 5 mL x 2 bottle Note Use Pretreat 3 with Cultured Adherent Cells and Peripheral Blood Mononuclear Cells PBMC Use Pretreat 4 for Fresh Frozen Tissues 6 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue AED CELL DIAGNOSTI IMPORTANT RNAscope Reagent Kits share the same Pretreatment Kit and Wash Buffer but have unique Detection Kits Do not interchange the reagent components of the Detection Kits even those having the same name 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 this 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 availa
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