320513_FreshFrozen SamplePrep Part1_UM_11052015
Contents
1. Proceed to the RNAscope Assay While slides are incubating proceed immediately to the RNAscope Multiplex Fluorescent Detection using the RNAscope Multiplex Fluorescent Detection Kit User Manual PART 2 Document No 320293 Ensure you have the appropriate user manual User manuals are available at www acdbio com technical support user manuals 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 RNAscope Fluorescent Multiplex Detection using the RNAscope Multiplex Fluorescent Detection Kit User Manual PART 2 Document No 320293 Troubleshooting For troubleshooting information please contact technical support at support acdbio com 14 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue ADVANCED CELL DIAGNOSTICS 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 hyrophobic Recommended Recommended Relative template size barrier in number of drops volume per slide per slide pL 7 i E 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 0 752. Workflow Fix the sections 15 MIN 4 Dehydrate the sections 20 MIN 4 Ootional Stopping Point 2 Create a hydrophobic barrier 10 MIN Apply Protease IV 30 MIN 4 Proceed IMMEDIATELY to the RNAscope Assay Immediately proceed to the RNAscope Fluorescent Multiplex Detection Kit Part 2 User Manual Document No 320293 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue 11 ies y j i i L k rit 4 ADVANCED CELL DIAGNOSTICS Materials required Fix the sections Dehydrate the sections 1X PBS 10 neutral buffered formalin NBF 4 paraformaldehyde 100 ethanol Tissue Tek Vertical 24 Slide Rack Tissue Tek Staining Dishes 5 required ImmEdge Hydrophobic Barrier Pen Chill 200 mL 10 NBF fresh 10 NBF or 4 paraformaldehyde 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 mmediately immerse slides in the pre chilled fixative Fix for 15 MIN at 4 C Note Formalin that has been stored for more than six months exposed to air for more than a week or used repeatedly may result in suboptimal tissue fixation Reagents may be prepared ahead of time Ensure all containers remain covered l oo ao 2 Prepare 200 ml 50 ethanol 200 ml 70 ethanol and 600 ml 100 ethanol in Tissue Tek Staining Dishes Place the slides in 50 ethanol for 5 MIN at ROOM TEMPERATURE RT Place the slides in 70 etha
3. 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 N 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 cryoembedding 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 surtace of liquid nitrogen or isopentane cooled by dry ice or liquid nitrogen or place the cryomold on dry ice A Store the frozen block in an airtight 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 2 Cut 10 20 pm sections and mount onto SUPERFROST PLUS SLIDES 10 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue ADVANCED CELL DIAGNOSTICS 3 Keep the sections at 20 C to dry 4 Store the sections in slide boxes wrapped airtight with aluminum foil or ziplock 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 Use sectioned tissue within 3 months Prepare the slides for the RNAscope Assay
4. 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 Fresh Frozen Tissue 15 ADVANCED CELL DIAGNOSTICS Chemical safety ZN Appendix B Safety WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general satety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets SDSs provided before you store handle or work with any chemicals or hazardous materials To obtain SDSs http www acdbio com technical support user manuals Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals tor 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 alll local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Bi
5. 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 olex 2 plex multiplex and automated assays are all available The RNAscope Assay procedure is illustrated in Figure l 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 pipettetree worktlow 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 rormalintixed parattinembedded FFPE tissues fresh frozen tissues fixed frozen tissues tissue microarray TMA and cell samples Visit www acdbio com and contact support acdhio com for more information Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue 5 ALD ADVANCED CELL DIAGNOSTICS Figure 1 Procedure overview ZZ Target RNA Specific Oligo Probes Se PreAMP AMP Label Probe Z Ze 2 pm zZ z z Zz iz zz z zZ AS se mRNA _ a MRNA 1 Tissue section 2 Hybridize to target RNA 3 Amplify signal Start with properly Hybridize multiple sets of Use up to fo
6. with your local lab supplier 8 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue ADVANCED CELL DIAGNOSTICS P Chapter 2 Before You Begin Prior to running the RNAscope Assay on your samples for the first time we recommend thot you e View the video demonstrations available at http www acdbio com technical support learn more 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 16 for more information Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue 9 ADVANCED CELL DIAGNOSTICS 3 Chapter 3 Prepare and Pretreat Samples Fresh frozen sample preparation and pretreatment are described in the following protocol For other sample types and preparation methods contact support acdhio com for the latest protocols and guidelines IMPORTANT We highly recommend following these guidelines Wwe cannot guarantee assay results with other preparation methods Prepare fresh frozen sections Materials required e Scalpel e Forceps Cryo embedding medium OCT Dry ice liquid nitrogen
7. 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 found on the ADC website at www acdbio com technical support user manuals f you have any questions please contact Advanced Cell Diagnostics at www acdbio com about contact 18 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue Headquarters emi 3960 Point Eden Way Hayward CA 94545 Phone 1 510 576 8800 Toll Free 1 877 576 3636 i we For support email support acdbio com ADVANCED CELL DIAGNOSTICS www acdbio com
8. Contents Chapter 1 Product Information cccccsseccceseceeescseuseceeeseeeanes 5 About this QUuide cccccccccsesssssceeeeeeeeeeeeceeeeeeeeeeeeetesssssseaeeeeeeeeeeeeeeeeeeeees 5 Product description cccccccccccceeeeesessseeeeeeeeeeeeeeeeececeseeeeeeesssssssaaaaeeeeeeeees 5 BIG IE OU NG OPE E E E A A nomad aaansnee 5 Sample y PES en Ree a E ee eee 5 RNAscope Assay reagents cccccccsssssssseeeeeeeeeceeeeeeceeeeeassssaeeeeeeeeeseeeeees 6 RNAscope Reagent Kit cccccccccssssssseseeeeeeeeeeeceeeeeeeeessssssaeeeeeeeeeeeeeeees 6 Required materials and equipment cccccccccccccecesceceeeeeenseessssssssasaeeeeeeeees 7 HybEZ Hybridization System cccccccccccccceeeeseeeeesessstsssssseaeeeeeeeeeeeeeeeens 7 User supplied materials ccccccccccccccccccccccccececceeeeseessestsssssseeeeeeeeseeees 7 Chapter 2 Before You Begin ccccsseccccssecceeeeceeeeeeceeaeeeeeeas 9 Important procedural guidelines cccccccccssccccecececeeeceeeeseeeeeeestsstssssaaeeeeees 9 Chapter 3 Prepare and Pretreat Samples sscccsseceeeeceees 10 Prepare fresh frozen Sections cccccccsssssscceeeccecceesssssssceeeeseceesesssttsneeeeees 10 Materials required cccccccccccccssssecececeeeceesseesssseeeeeceeceeessssseeeeeeeeeeeeaas 10 Prepare the IOC I cansecerysrassawenssnsic tntusioresatsnisemuchaarne nw saneedsnisomas er outinnsrentiatd 10 DSC NOM Me OCG sisirin ren
9. LY setesccssxsicecsadescwavvresver setenv sedaseysteedouetebunerenmreieeceeds 16 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue Documentation and Support ccccccssscccesececscceeeeeeseeeeeaeeees 18 Obtaining MSDS acta tic neern E AAE TAE O O 18 OB OO ea E AEE E E EE ET 18 Contact informatio sssesssrinsssnininr neei eiia A EES EN ANNEES 18 Limited product WGI GIN cccacscen conn dasetiee nena tanasar ne ueenntnenatereandx 18 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue ADVANCED CELL DIAGNOSTICS Chapter 1 Product Information Before using this product read and understand the information in Appendix B Safety on page 16 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 jn 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
10. USER MANUAL Af ADVANCED CELL DIAGNOSTICS RNAscope Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue using RNAscope Fluorescent Multiplex Assay PART 1 Document Number 320513 Compatible with the RNAscope Fluorescent Multiplex Detection Kit User Manual Part 2 see Document Number 320293 For Research Use Only RUO Not for Diagnostic Use Rev Date 11052015 For Research Use Only RUO Not for Diagnostic Use Trademarks RNAscope and HybEZ are trademarks of Advanced Cell Diagnostics Inc All other trademarks belong to their respective Owners Citing RNAscope 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 LC Bui S Nielson A Wu X Vo HT Ma XJ and Luo Y RNAscope A Novel In Situ RNA Analysis Platform for FormalinFixed ParattinEmbedded 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 2015 Advanced Cell Diagnostics Inc All rights reserved AED
11. al guidelines and legislation on biohazard and biosatety 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 csr_lyo 2004 11 en Intormation about the Registration Evaluation Authorisation and Restriction ot Chemicals REACH can be found at eur lex europa eu LexUriServ LexU riServ do uri 0J L 2010 133 0001 0043 EN PDF Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue 17 j i m ADVANCED CELL DIAGNOSTICS Documentation and Support Obtaining MSDSs Satety Data Sheets SDSs are available at www acdbio com technical support user manuals 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 technical sup port user manuals 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
12. ean eE iar seacneaauesearine 10 Prepare the slides for the RNAscope Assay cccceeessseeeeceeeeeeessssssaeeeees 11 VY OUI OY oree ER E E eonitorvienitenexensies NOE unensaee 11 Moterials required asaccccncesaniateaaeseacins esasasaomeasseniecsaaaueesenreniesanieieianeaae 12 PICMG GOCHONS uaipe riei EE A EE EAE 12 Dehydrate the sections c cccccccccccccccceeesceseeeseseesnstssseeeeeeeeeeeeeeeeees 12 Create a hydrophobic barrier cccccccessssscecceecceeceeeceeeeeseeeeeeeenntnaas 12 Prereat SGIMPIES csnsencsasanenonsasiacssanniaseqabeernentntencsaonneneanbrignananiaseeaeeteneemanes 13 Materials MECUING Clear pececerexcuecossearecevaceiaxna eakesndeacectseemeseeteaeeeneraredenteane 13 Prepare the equipment 0 0 0 ccccccessseceeccceeeeessssssseeeeeeeesesessssseeeeeeeeeees 13 Apply Prolease V sajna coccinea anteater eens ee eee ee 13 Proceed to the RNAscope Assay cccccccessssscceeeceeeeessssseeeeeeeeeeeeesesssaeeeees 14 T5BTroubleshooting ccccccccccccccccssssseceeccccceeesesssseeeeeeceeseeessseeeeeeeeseeensaaas 14 Appendix A Reagent Volume Guidelines cccccseceeneceeeees 15 Determine reagent volume cccceeeeeessssceeeeeeeeeceeeeeeceeeeeeeetessssssaaaeeeees 15 Appendix B Safety cccccccccescccesscceesecceeeceeaeeceeeeeeeaneeeeaees 16 Chemical SE PY ee oa sersca Serensenecaice smi aanceencasint on cosne sade sanoenncecocemee ev amaecanemessseeia 16 Biological hazard SGlE
13. ife of nine months from the date of bulk manufacture when stored as indicated in the following table Pretreatment Kit Cat No 320842 Protease IV 2X Ready To Use RTU protease 4 5 mL x 2 bottle Protease III 1X Ready To Use RTU protease 4 5 mL x 1 bottle Note Use Protease III with Cultured Adherent Cells and Peripheral Blood Mononuclear Cells PBMC Use Protease IV for Fresh Frozen Tissues 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 6 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue ADVANCED CELL DIAGNOSTICS 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 available at www acdbio com technical support user manuals and view the training video at www acdbio com technical support lear
14. n more The system contains the following components 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 100 ethanol ALREAGAL Tissue Tek Vertical 24 Slide Rack American Master Tech Scientific MLS LWSRA24 Tissue Tek Staining Dish 6 required American Master Tech Scientific MLS LWT4457EA Tissue Tek Clearing Agent Dish xylene resistant 3 American Master Tech Scientific MLS required ProLong Gold Antifade Reagent Life Technologies P36930 Cover glass 24 x 50 mm Fisher Scientific MLS 12 545 F 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 pL MLS Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue 7 ADVANCED CELL DIAGNOSTICS EJ Description Supplier Cat No U ML Distilled water Tubes various sizes MLS Fume hood MLS Graduated cylinder MLS Parafilm MLS Paper towel or absorbent paper MLS Glass beaker 1 or 2 L ML Hot plate Fisher Scientific MLS Aluminum foil MLS U 300 49SHP Forceps large MLS Thermometer MLS 20 bleach MLS Microscope and accessories MLS Major Laboratory Supplier in North America For other regions please check Catalog Numbers
15. nol for 5 MIN at RT Place the slides in 100 ethanol for 5 MIN at RT Repeat step 6 with tresh 100 ethanol OPTIONAL STOPPING POINT 2 Slides may be stored in 100 ethanol at 20 C for up to 1 WEEK Prolonged storage may degrade sample RNA Create a hydrophobic barrier I Take slides out of 100 ethanol 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 Inmedge hydrophobic barrier pen See example below size of this hydrophobic barrier is OFS 079 Note Refer to Appendix A Reagent Volume Guidelines on page 15 to determine the recommended number of drops needed per slide IMPORTANT Do not let the barrier touch the section The 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 rY d 1 rt 1 p D p r p B he g a I r ie ADVANCED CELL DIAGNOSTICS Hydrophobic Barrier 0 75 n q e UTE oe Tissue Note VVe 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 f you need to reapply the hydrophobic barrier during the following procedures dry the appropriate area of the slide
16. ological hazard safety AN 16 WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit intectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear tace 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 Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue In the U S In the EU ADVANCED CELL DIAGNOSTICS U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdegov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara dr waisidx_01 2029cr1910a_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 cdegov Check loc
17. ur signal amplification systems Visualize target prepared tissue sections gene specific probe pairs to to detect multiple target RNAs Probes are RNA using a and pretreat to allow target mRNAs hybridized to a cascade of signal standard fluorescent access to target RNA amplification molecules culminating in microscope binding of dye labeled probes visible in different fluorescent channels 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 RNAscope 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 tind a gene specific Target Probe Visit http www acdbio com control slides and probes to order appropriate Control Probes RNAscope Reagent Kit Each RNAscope Reagent 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 subkits including a Pretreatment Kit which come with up to three reagents Pretreatment of Fresh Frozen tissue Cat No 322340 requires only Protease IV Pretreatment Kit instructions are provided in this guide For information on the other subkits and directions for use refer to an RNAscope Assay User Manual The reagents have a shelf l
18. with a Kimwipe Do not touch the tissue section Pretreat samples IMPORTANT Do not apply RNAscope Hydrogen Peroxide or RNAscope Target Retrieval 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 Protease Ill amp IV Prepared slides Distilled water e Paper towel or absorbent paper e HybEZ Humidifying System Tissue Tek Slide Rack Tissue Tek Staining Dish Prepare the equipment e Turn on HybEZ Oven and set temperature to 40 C e Place a Humiditying Paper in the Humidity Control Tray and wet completely with distilled water Apply Protease IV IMPORTANT View the wash step video at www acdbio com technical support learn more before proceeding 1 Place the dried slides on the HybEZ Slide Rack and add 5 drops of Protease IV to entirely cover each section 2 Incubate for 30 MIN at RT Sample Preparation and Pretreatment Guide for Fresh Frozen Tissue 13 r q d iy A g i4 ADVANCED CELL DIAGNOSTICS 3 Take each slide one at a time from the HybEZ Slide Rack and tap and or flick to remove the excess liquid mmediately 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
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