RNAscope® 2.5 HD Detection Reagent – RED User Manual
Contents
1.2. RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 ADVANCED CELL DIAGNOSTICS Required materials and equipment The following materials and equipment are needed to perform the RNAscope 2 5 Assay HybEZ Hybridization System IMPORTANT The RNAscope 2 5 Assay has been verified 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 2 5 Assays Incubation steps in the RNAscope 2 5 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 http www acdbio com documents support documents and view the training video at http www acdbio com technical support learn more The system contains the following components T 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 EcoMount listed in the following table T Description Su
3. Fume hood e RNAscope 2 5 AMP 4 e Xylene e RNAscope 2 5 AMP 5 RED e Tissue Tek Staining Dish 3 e RNAscope 2 5 AMP 6 RED e Tissue Tek Clearing Agent Dish e RNAscope 2 5 Fast RED A xylene resistant 1 amp RNAscope 2 5 Fast RED B e Gill s Hematoxylin e Ammonium hydroxide 28 30 e Graduated cylinder e Parafilm e HybEZ Humidifying System e Water bath or incubator e Tissue Tek Vertical 24 Slide Rack e Tubes various sizes e Paper towel or absorbent paper e Pipettors and tips 1 1000 pl e Dry oven e EcoMount e Cover Glass 24 mm x 50 mm Prepare the materials You may prepare the reagents at the same time you prepare pretreatment reagents Refer to a sample preparation and pretreatment user manual available at http www acdbio com technical support user manuals Some of the materials may be prepared in advance and stored at room temperature Prepare 1X Wash Buffer 1 Prepare 3 Lof 1X Wash Buffer by adding 2 94 L distilled water and 1 bottle 60 ml of RNAscope Wash Buffer SOX to a large carboy Mix well Note VVarm RNAscope 50X Wash Buffer up to 40 C for 10 20 MIN before preparation 1X Wash Buffer may be prepared ahead of time and stored at room temperature for up to one month Prepare counterstaining reagents 1 In the fume hood prepare 50 Hematoxylin staining solution by adding 100 ml Gill s Hematoxylin to 100 ml distilled water in a
4. all necessary safety procedures Refer to Appendix C Safety on page 21 for more information RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 ADVANCED CELL DIAGNOSTICS 3 Chapter 3 RNAscope 2 5 Assay IMPORTANT For Part 1 Formalin Fixed ParaffinEmbedded FFPE Sample Preparation and Pretreatment User Manual see Document No 322452 USM This procedure flows directly from sample preparation and pretreatment Refer to the appropriate sample preparation and pretreatment user manual for your specific sample type Workflow Prepare the materials 10 30 MIN 4 Run the assay 4 HRS 45 MIN Hybridize probe 2 HRS Hybridize Amp 1 30 MIN Hybridize Amp 2 15 MIN Hybridize Amp 3 30 MIN Hybridize Amp 4 15 MIN Hybridize Amp 5 30 MIN Hybridize Amp 6 15 MIN Detect the signal 10 MIN Counterstain the slides 2 MIN Mount samples 5 MIN Evaluate the samples RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 ADVANCED CELL DIAGNOSTICS Materials required for the assay Materials provided by Materials provided by Other Materials and Equipment RNAscope 2 5 HD Detection Kit RED RNAscope Probes e RNAscope Wash Buffer 50X e Target Probe e Prepared sections e RNAscope 2 5 AMP I e Positive Control Probe e Distilled water e RNAscope 2 5 AMP 2 e Negative Control Probe e Carboy gt 3L e RNAscope 2 5 AMP 3 e
5. e Assess negative control background One dot to every 10 cells displaying background DAB 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 can enhance the value of in situ hybridization results by enabling a semi quantitative scoring guideline utilizing the estimated number of punctate dots present within each cell boundary An example on 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 If your gene expression level is higher or lower than this range you may need to scale the criteria accordingly Categorize staining into five grades O 1 2 3 and 4 according to the following table Staining Microscope Objective Scoring Score 0 No staining or less than 1 dot to every 10 cells 40X magnification 1 3 dots cell visible at 20 40X magnification 2 4 10 dots cell 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 RNAscope 2 5 HD Detection Reagent RED User Manual 322360 U
6. ADVANCED CELL DIAGNOSTICS RNAscope 2 5 HD Detection Reagent RED User Manual PART 2 Document Number 322360 USM For Part I Sample Preparation Pretreatment Guide for Formalin Fixed Parattin Embedded FFPE For RNAscope 2 5 Assay see Document Number 322452 USM For Research Use Only RUO Not for diagnostic use 322360 USM 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 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 Paraftin 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 2015 Advanced Cell Diagnostics Inc All rights reserved RNAscope 2 5 HD Detection Reagent R
7. ED User Manual 322360 USM Rev Date 11052015 ADVANCED CELL DIAGNOSTICS Contents Chapter 1 Product Information cscccsccssesscsssessesseessesseeseeeseenees 5 Abo tthis guide JE ee eee eT EEEE 5 Product description se22cse25ecscneesesscscesneecacaansedacnaesedaescaseseessnsneaessesedaczanandasanexins 5 Bete Ca Io DIe PE E E cathe hcnbhehbbchbhehibcnbheablannhen cher 5 NA 5 Kit contents and storage uvvsrseapsaseseseplenpasnGmbumgemuete 6 RNAscope NN 6 RNAscope 2 5 HD Reagent Kit RED csssssssesssesssssnsssssnssnsenes 7 Required materials and eqUiPMeENt cccccccccscseesesseseesesecsesscsessesecseeseseeseseees 8 HybEZ Hybridization System icci senisiaseccisinceeisinteisintenndeeieticcaeneds 8 User supplied makro secacadeqcsacecase aveesceancensvesacensieenteantsesdesadeesseanCenteesect 8 Chapter 2 Before You Begin csccsccsscsseeseeseeeseeseesseeseeseeseeens 10 Important procedural guidelines cccccccccssessesesseseesessesesseseeesseeseseeseeeenes 10 Chapter 3 RNAscope 2 5 Assay ccccscssscsseessesseseesseeseeseeeneees 11 Va aia aenmaeeaemnennetes esas 11 Materials required for the assay sexccocecelaceecsesesesadeleseaciesetesuseseeseesdieiesadescantes 12 Prepare the MIATA GINS ode Seoc acnapssastseanSnasdcocdsan consaastcosnastnasSaarinesseavtserteoseens 12 Prepare 1X Wash ber Vav4v4v4v4vv4v47 esse 12 Prepare counterstaining reagents esccccesssseecesssseeeesss
8. NT For Part 1 Formalin Fixed ParaffinEmbedded FFPE Sample Preparation and Pretreatment User Manual see Document No 322452 USM Prior to running the RNAscope 2 0 Assay on your samples for the first time we recommend that you View the video demonstrations available at http www acdbio com technical support learn more 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 Start with properly fixed and prepared sections Refer to Appendix A Tissue Pretreatment Recommendation on page 18 and to our sample preparation and pretreatment user guides available at http www acdbio com technical support user manuals Use only samples mounted on SuperFrost Plus Slides Fisher Scientific Cat No 12 550 15 Follow the recommended pretreatment guidelines for your sample Refer to our sample preparation and pretreatment user guides available at http www acdbio com technical support user manuals Always run positive and negative control probes on your sample to assess sample RNA quality and optimal permeabilization Do not substitute required materials Assay has been validated with these materials only Follow the protocol exactly for best results Do not let your sections dry out during the procedure Use good laboratory practices and follow
9. Rev Date 11052015 Net Mee ADVANCED CELL DIAGNOSTICS 3 Remove the HybEZ Control Tray from the oven and remove HybEZ Slide Rack 4 One slide at a time quickly remove excess liquid and place slide in a TissueTek Slide Rack submerged in the Tissue Tek Staining Dish filled with 1X Wash Buffer 5 Wash slides in 1X Wash Buffer for 2 MIN at RT with occasional agitation 6 Repeat Step 5 with fresh 1X Wash Buffer Hybridize AMP 2 1 Take each slide one at a time from the TissueTek Slide Rack and tap and or flick to remove the excess liquid from slides Place slides in the HybEZ Slide Rack in the HybEZ Humidity Control Tray Add 4 drops of AMP 2 to entirely cover each section 2 Close tray and insert into the oven for 15 MIN at 40 C 3 Remove the HybEZ Control Tray from the oven and remove HybEZ Slide Rack 4 One slide at a time quickly remove excess liquid and place slide in a TissueTek Slide Rack submerged in the Tissue Tek Staining Dish filled with 1X Wash Buffer 5 Wash slides in 1X Wash Buffer for 2 MIN at RT with occasional agitation 6 Repeat Step 5 with fresh 1X Wash Buffer Hybridize AMP 3 1 Take each slide one at a time from the TissueTek Slide Rack and tap and or flick to remove the excess liquid from slides Place slides in the HybEZ Slide Rack in the HybEZ Humidity Control Tray Add 4 drops of AMP 3 to entirely cover each section 2 Close tray a
10. SM Rev Date 11052015 AED ADVANCED CELL DIAGNOSTICS Quantitative image analysis RNAscope Spot Studio Software is designed for pathologists with no prior training in image analysis This intuitive software allows users to obtain 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 Control examples Figure 2 is an example of human liver cancer sections using dapB Negative Control Probe and PPIB Positive Control Probe at 40X magnification Figure 2 RNAscope 2 5 HD Detection Kit RED performed on human liver cancer sections using the dapB Negative Control Probe Cat No 310043 and PPIB Positive Control Probe Cat No 313901 40X magnification 2 b Hs PPIB Troubleshooting For troubleshooting information please contact technical support at support acdbio com RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 ADVANCED CELL DIAGNOSTICS A 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 Samples prepared differently than the sample preparation protocol found in Part I Formalin Fixed P
11. 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 Hydrophobic Recommended Number Recommended Volume Relative Template Size Barrier in of Drops per Slide per Slide pl 0 75 x 0 75 t A 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 RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 20 ADVANCED CELL DIAGNOSTICS C Appendix C Safety Chemical safety 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 Safety Data Sheets SDSs before you store handle or work with any chemicals or hazardous materials To obtain SDSs see http www acdbio com technical support user manuals e Minimize contact with chemicals Wear appropriate personal protective equipmen
12. arattin Embedded FFPE Sample Preparation and Pretreatment User Manual Document No 322452 USM Tissue pretreatment recommendation 1 Stain representative samples using the positive and negative control probes 2 Fix sample in fresh 10 NBF for 16 32 HRS at RT 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 amount of time for the RNAscope Target Retrieval Reagents and or RNAscope Protease Plus Reagent Mild Standard Extended RNAscope Target Retrieval Reagents 15 MIN 15 MIN 15 30 MIN RNAscope Protease Plus 15 MIN 30 MIN 30 MIN Note Sample types such as certain Xenografts and Cell Pellets require less time For thes tissue types vary the RNAscope Target Retrieval Reagents time to 8 MIN and RNAscope Protease Plus time to 15 MIN 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 10 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 Intesti
13. ay has additional amplification steps that allow observable results under 10 20X magnification RNAscope 2 5 Assays offer the choice of two Detection Reagents Brown DAB and Red Fast Red which enable RNA molecules to be visualized as brown or red chromogenic dots respectively RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 ASD ADVANCED CELL DIAGNOSTICS Figure 1 Procedure overview re Oo ZZ Target RNA Specific Oligo Probes PreAMP AMP Label Probe 7358 1 Tissue section 2 Hybridize to target RNA 3 Amplify signal Start with properly Hybridize gene specific probe Probes are hybridized to a cascade of Visualize target prepared sections and pairs to the target mRNA signal amplification molecules culminating RNA using a pretreat to allow access fo in binding of HRP or AP labeled probes standard bright target RNA The 2 5 Assay enhances signal further with field additional amplification steps Add DAB or microscope Fast Red substrate to detect target RNA Kit contents and storage The RNAscope 2 5 Assay requires the RNAscope Probes and the RNAscope Detection Kit Probes and Detection Kits are available separately RNAscope Probes The RNAscope Probes consist of the user specified Target Probe and the Positive and Negative Control Probes Visit www acdbio com products target probes search produd to find a gene specific Target Probe Visit http w
14. e purple 2 Immediately transfer the slide rack back into the staining dish containing tap water and wash slides 3 5 times by moving the rack up and down Keep repeating with fresh tap water until the slides are clear while sections remain purple RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 ADVANCED CELL DIAGNOSTICS 3 Replace tap water in the staining dish with 0 02 Ammonia water Move rack up and down 2 3 times Section should tum blue 4 Replace Ammonia water with tap water Wash slides 3 5 times Mount the samples 1 Remove the slide rack from the staining dish and dry slides in a 60 dry oven for at least 15 MIN until slides completely dry IMPORTANT The RED substrate is alcohol sensitive Do not dehydrate the slides in alcohol 2 Briefly dip one slide into fresh pure xylene and immediately place 1 2 drops of EcoMount on the slide before the xylene dries IMPORTANT Use the EcoMount mounting medium only 3 Carefully place a 24 mm x 50 mm coverslip over the tissue section Avoid trapping air bubbles 4 Repeat steps 2 and 3 for each slide 5 Air dry slides for gt 5 MIN Evaluate the samples 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 cells at 20 40X magnification
15. ed by Advanced Cell Diagnostics contact the chemical manufacturer Obtaining support For the latest services and support information go to http www acdbio com technical support support overview At the website you can Access telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Search for user documents SDSs application notes citations training videos and other product support documents 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 found on the ACD website at http www acdbio com store terms f you have any questions please contact Advanced Cell Diagnostics at http www acdbio com about contact RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 23 Headquarters 3960 Point Eden Way Hayward CA 94545 Phone 1 510 576 8800 Toll Free 1 877 576 3636 AL D A For support email support acdbio com ADVANCED CELL DIAGNOSTICS www acdbio com
16. ely cover each section RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 L TEN F AG gd Fr MN S tuss ADVANCED CELL DIAGNOSTICS 2 Place the HybEZ Slide Rack in the HybEZ Humidity Control Tray Seal tray and incubate for 30 MIN ot RT 3 Remove the HybEZ Slide Rack from the HybEZ Humidity Control Tray 4 One slide at a time quickly remove excess liquid and place slide in a TissueTek Slide Rack submerged in the Tissue Tek Staining Dish filled with 1X Wash Buffer 5 Wash slides in 1X Wash Buffer for 2 MIN at RT with occasional agitation 6 Repeat Step 5 with fresh 1X Wash Buffer Hybridize AMP 6 1 Take each slide one at a time from the TissueTek Slide Rack and tap and or flick to remove the excess liquid before placing in the HybEZ Slide Rack Add 4 drops of AMP 6 to entirely cover each section 2 Place the HybEZ Slide Rack with the slides in the HybEZ Humidity Control Tray Close tray and incubate for 15 MIN at RT 3 Remove the HybEZ Slide Rack from the HybEZ Humidity Control Tray 4 One slide at a time quickly remove excess liquid and place slide in a TissueTek Slide Rack submerged in the Tissue Tek Staining Dish filled with 1X Wash Buffer 5 Wash slides in 1X Wash Buffer for 2 MIN at RT with occasional agitation 6 Repeat Step 5 with fresh 1X Wash Buffer IMPORTANT Staining intensity can be modified by adjustin
17. g the AMP 5 incubation time Detect the signal 1 Briefly spin down the contents of the Fast RED B tube to be sure content is at the bottom of the tube before opening the cap 2 Depending on the size of your hydrophobic barrier prepare sufficient RED working solution per section by using a 1 60 ratio of Fast REDB to Fast RED A For example for a 0 75 x 0 75 barrier add 2 pl of Red B to 120 pl of Red A into a tube Mix well IMPORTANT Use the Fast RED B solution within 5 MIN Do not expose to direct sunlight or UV light 3 Take each slide one at a time from the Tissue Tek Slide Rack and tap and or flick to remove the excess liquid before placing in the HybEZ Slide Rack 4 Pipette 120 pL RED solution onto each tissue section Ensure sections are covered 5 Place the HybEZ Slide Rack with the slides in the HybEZ Humidity Control Tray Seal tray and incubate for 10 MIN at RT 6 Remove the HybEZ Slide Rack from the HybEZ Humidity Control Tray 7 To remove the RED working solution from the slides tilt each slide one at a time over a waste Counterstain the slides l container and tap the corner on the edge of the container mmealately insert the slide into a Tissue Tek Slide Rack submerged in a Tissue Tek Staining Dish filled with tap water Rinse again with fresh tap water Move the Tissue Tek Slide Rack into the staining dish containing 50 Hematoxylin staining solution for 2 MIN at RT Slides will b
18. i acces acaceseaelecucereleleceeuieracenteenaceesesutesateanieetiaasades 20 Appendix C SONNY sii cicasssnecsssramnccniassasistirersinncnaimemniesvene 21 el saa ae ates yaaa cates ea as ce eeees teas eas nee gens eas eo 21 miie EU FRERE EE T 22 Documentation and Support ccsccsccssscssescessessseseeseesseeseeeseenes 23 oolite s E REE 23 Obtaining SSS cat pera cut tcscnthctcncath ck spect chenptthahonenthahenemthahshentlah shen bakini iiiki 23 Contact information ERR EE BROR EEE EEE eects 23 Limited product varankrssssesoessednuekemedentkenn hava 23 RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 ADVANCED CELL DIAGNOSTICS Chapter 1 Product Information Before using this product read and understand the safety information in Appendix C Safety on page 21 IMPORTANT VVe recommend reading the entire user manual before beginning any protocols About this guide This user manual provides guidelines and protocols to use the RNAscope 2 5 HD Detection Kit RED Cat No 322360 RNAscope Assays are compatible with a variety of sample types You must use both an RNAscope Detection Kit user manual and a Sample Preparation and Pretreatment user manual to perform the entire assay IMPORTANT For Part 1 Formalin Fixed ParaffinEmbedded FFPE Sample Preparation and Pretreatment User Manual see Document No 322452 USM Visit www acdbio com technical support user manual
19. ing 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 RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 21 ADVANCED CELL DIAGNOSTICS e Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR81910 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 infectious materials e Additional information about biohazard guidelines is available at www cde 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 at http 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 http echa europa eu regulations reach RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 22 ADVANCED CELL DIAGNOSTICS Obtaining SDSs Documentation and Support Safety Data Sheets SDSs are available at http www acdbio com technical support user manuals For the SDSs of chemicals not distribut
20. luded in separate sample preparation and pretreatment user manuals The reagents have a shelf life of nine months from the date of bulk manufacturing when stored as indicated in the following table Pretreatment Reagents Cat No 322300 and 322000 k Reagent Quantity Storage e RNAscope Hydrogen Peroxide e RNAscope Protease Plus e 3 mL x 2 bottles e 4 5 mL x 1 bottle 2 8 C RNAscope Target Retrieval 10X 70 mL x 4 bottles Room temp 15 30 C RNAscope 2 5 HD Detection Reagents RED Cat No 322360 A Reagent Quantity Storage RNAscope 2 5 AMP 1 3 mL x 1 bottle 2 8 C RNAscope 2 5 AMP 2 4 5 mL x 1 bottle 2 8 C RNAscope 2 5 AMP 3 3 mL x 1 bottle 2 8 C RNAscope 2 5 AMP 4 4 5 mL x 1 bottle 2 8 C RNAscope 2 5 AMP 5 RED 4 5 mL x 1 bottle 2 8 C RNAscope 2 5 AMP 6 RED 3 mL x 1 bottle 2 8 C RNAscope 2 5 Fast RED A 3 mL x 1 bottle 2 8 C RNAscope 2 5 Fast RED B 50 pl x 1 vial 2 8 C RNAscope Wash Buffer Reagents Cat No 310091 rv Reagent Quantity Storage RNAscope Wash Buffer 50X 60 mL x 4 bottles Room temp 15 30 C IMPORTANT RNAscope HD RED and BROWN Reagent Kits share the same Pretreatment Reagents Hydrogen Peroxide Target Retrieval and Protease Plus and Wash Buffer but have unique Detection Reagents Do not interchange the reagent components of the Detection Kits even though they have the same name
21. n prior fo use 1 Tap and or flick to remove excess liquid from slides Place slides in the HybEZ Slide Rack located in the HybEZ Humidity Control Tray Add 4 drops of the appropriate probe to entirely cover each section Note Refer to Appendix B Reagent Volume Guidelines on page 20 to determine the recommended number of drops needed per slide For example for a 0 75 x 0 75 barrier add 4 drops of the appropriate probe 2 Cover the HybEZ Humidity Control Tray with lid and insert into the oven for 2 HRS at 40 C IMPORTANT To prevent evaporation make sure the turn nob is completely turned to lock position 3 Remove the HybEZ Control Tray from the oven and remove HybEZ Slide Rack 4 One slide at a time quickly remove excess liquid and place slide in a TissueTek Slide Rack submerged in the Tissue Tek Staining Dish filled with 1X Wash Buffer 5 Wash slides in 1X Wash Buffer for 2 MIN at RT Agitate slides by moving the slide rack up and down in the dish 6 Repeat Step 5 with fresh 1X Wash Buffer Hybridize AMP 1 1 Take each slide one at a time from the Tissue Tek Slide Rack and tap and or flick to remove the excess liquid from slides Place slides in the HybEZ Slide Rack in the HybEZ Humidity Control Tray Add 4 drops of AMP 1 to entirely cover each section 2 Close tray and insert into the oven for 30 MIN at 40 C RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM
22. nd insert into the oven for 30 MIN at 40 C 3 Remove the HybEZ Control Tray from the oven and remove HybEZ Slide Rack 4 One slide at a time quickly remove excess liquid and place slide in a Tissue Tek Slide Rack submerged in the Tissue Tek Staining Dish filled with 1X Wash Buffer 5 Wash slides in 1X Wash Buffer for 2 MIN at RT with occasional agitation 6 Repeat Step 5 with fresh 1X Wash Buffer Hybridize AMP 4 1 Take each slide one at a time from the TissueTek Slide Rack and tap and or flick to remove the excess liquid from slides Place slides in the HybEZ Slide Rack in the HybEZ Humidity Control Tray Add 4 drops of AMP 4 to entirely cover each section 2 Place the HybEZ Slide Rack in the HybEZ Humidity Control Tray Close tray and insert into the oven for 15 MIN at 40 C 3 Remove the HybEZ Control Tray from the oven and remove HybEZ Slide Rack IMPORTANT Do not insert tray into the HybEZ Oven for the rest of the procedure 4 One slide at a time quickly remove excess liquid and place slide in a TissueTek Slide Rack submerged in the Tissue Tek Staining Dish filled with 1X Wash Buffer 5 Wash slides in 1X Wash Buffer for 2 MIN at RT with occasional agitation 6 Repeat Step 5 with fresh 1X Wash Buffer Hybridize AMP 5 1 Take each slide one at a time from the TissueTek Slide Rack and tap and or flick to remove the excess liquid before placing in the HybEZ Slide Rack Add 4 drops of AMP 5 to entir
23. ne Tumor Standard Embryo Normal Standard Brain Normal Standard Spleen Normal Mild Eye Retina Normal Standard Mild Liver Normal Extended RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 ADVANCED CELL DIAGNOSTICS Species Tissue Type Pathology Pretreatment Condition Kidney Normal Standard Human Breast Tumor Standard Colon Tumor 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 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 G Mild HeLa cells fixed with 10 Standard Formaldehyde PBS ACD Control Tissue Microarray RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 ADVANCED CELL DIAGNOSTICS Appendix B Reagent
24. neeeeeseneeeesenaeees 12 Prepare mounting reagents essccesssnceceesssteecesssneeeeessneeeeeseneeeesenaaees 13 Equilibrate reagents ccccccccesssessescsseseeseseesesecseescsecsesecseesesesseeeesseseseens 13 RU TE SSO REDERE A A AAAA 13 Hybridize probe ch eetyacnenereeqareeeonteeeeeecanees 13 Fell vr 13 Hybridize igh an Ree nD OO TOO OO ET OO 14 Hybridize AMP 3 honsharclonchanohonetenalenstenalenstenalenshonalenchenalshabeaclshaheuctanetonte 14 le 14 Hybridize 0 EEE Eee 14 Hybridize NN 15 Detect hesignal s cc cucdhocucstceutccntceabhenrecebhearheehonucleshenabGrabaseharabecnhgeabearb cass 15 Counterstain the slides saninncnccnmnmancamenenannmmnenammaans 15 Moe mple 16 Evaluate he samples ssagasdasddeendsde ae 16 Scoring guidelines aescchopctcnshaneccnshenseinabenohenahenaruckennnenstanshcnehincbenetes stoned eats 16 Quontitative image analysis au mudsoedrtebntnnmihijjnthj 17 Control Fo ae oekte 17 keubleh onguruassassoseteatmetvetmsees freskene 17 RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 ADVANCED CELL DIAGNOSTICS Appendix A Tissue Pretreatment Recommendation c0sssseees 18 Tissue pretreatment recommendation ccccccscceesesesseeseseesesecseeseseeseeseseeeesees 18 Tissue specific pretreatment conditions cccccccseseeseseseteeseseteestseeeseseseeaes 18 Appendix B Reagent Volume Guidelines rrrnrrrnrrrnrrrnvrrnvrrnnnr 20 Determine reagent volume
25. pplier Cat No Gill s Hematoxylin l American Master Tech Scientific MLS HXGHE1LT Xylene Fisher Scientific MLS X3P 1GAL Tissue Tek Vertical 24 Slide Rack American Master Tech Scientific MLS LWSRA24 Tissue Tek Staining Dish 3 required American Master Tech Scientific MLS LWT4457EA lt ad Agent Dish xylene resistant American Master Tech Scientific MLS LWTA456EA 95 Ethanol EtOH American Master Tech Scientific ALREACS EcoMount required Biocare EM897L 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 Distilled water MLS Tubes various sizes MLS Fume hood MLS RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 ADVANCED CELL DIAGNOSTICS T Description Supplier Cat No Graduated cylinder MLS Parafilm MLS Paper towel or absorbent paper MLS Microcentrifuge MLS Microscope and accessories MLS Drying oven capable of holding temperature at MLS 60 1 C Major Laboratory Supplier in North America For other regions please check Catalog Numbers with your local lab supplier RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 ADVANCED CELL DIAGNOSTICS 2 Chapter 2 Before You Begin IMPORTA
26. s to download a sample preparation 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 RNAscope Assays do not require the RNAffr environment used for traditional ISH The assays are based on ACD s patented signal amplification and background suppression technology Compared with the RNAscope 2 0 Assay the 2 5 Assay incorporates an additional signal amplification step which enhances the signal for low expressing genes and RNA present in archived samples and partially degraded specimens Overview The RNAscope Assay procedure is illustrated in Figure I on page 6 The procedure can be completed in 7 8 hours or conveniently divided over two days Most of the RNAscope Assay reagents are available in convenient Ready To Use RTU dropper bottles and provide a simple nearly pipettetree workflow Starting with properly prepared tissue samples sections are first pretreated and then RNA specific probes are hybridized to target RNA The signal is amplified using a multistep process followed by hybridization to horseradish peroxidase HRP or alkaline phosphatase APHabeled probes and detected using a chromogenic substrate Each single RNA transcript appears as a distinct dot of chromogen precipitate visible using a common bright field microscope at 40 100X magnification The RNAscope 2 5 Ass
27. staining dish Note 50 Hematoxylin staining solution can be reused for up to 1 week RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 L TEN F AG gd Fr MN S tuss ADVANCED CELL DIAGNOSTICS 2 In the fume hood prepare 0 02 w v Ammonia water bluing reagent by adding 1 43 ml of IN Ammonium Hydroxide to 250 ml distilled water in a graduated cylinder or other container 3 Seal the cylinder with parafilm Mix well 3 5 times Note For assay quantitation it is critical to use Ammonium Hydroxide Prepare mounting reagents IMPORTANT Do not reuse deparaffinization reagents for dehydration of the slides after the assay 1 In the fume hood add 200 ml xylene to a clearing agent dish Note Reagents may be prepared ahead of time Ensure all containers remain covered Equilibrate reagents 1 Remove AMP 1 6 reagents from refrigerator and place at RT 2 Ensure HybEZ Oven and prepared Humidity Control Tray are at 40 3 Before each use warm the Target and or Control Probes for at least 10 MIN at 40 C in a water bath or incubator Swirl gently to mix Run the assay IMPORTANT Do NOT let sections dry out between incubation steps Work quickly and fill barrier with solutions IMPORTANT View the wash step video at http www acdbio com technical support learn more before proceeding Hybridize probe IMPORTANT Ensure probes are prewarmed to dissolve any precipitatio
28. t when handling chemicals for example safety 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 AN 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 materials Read and follow the applicable guidelines and or regulatory requirements in the follow
29. ww acdbio com control slides and probes to order appropriate Control Probes Each probe is sufficient for staining 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 The probes have a shelf life of two years from the date of bulk manufacturing when stored as indicated in the following table Target Probes vy Reagent Cat No Content Quantity Storage RNAscope Singleplex Target Probe Various Probe targeting specific RNA 3 mL x 1 bottle 2 8 C species gene Control Probes vy Reagent Cat No Content Quantity Storage RNAscope Positive Control Probe Various Probe targeting common 3 mL x 1 bottle 2 8 C species PPIB housekeeping gene RNAscope Negative Control Probe 310043 Probe targeting bacterial 3 mL x 1 bottle 2 8 C DapB gene dapB RNAscope 2 5 HD Detection Reagent RED User Manual 322360 USM Rev Date 11052015 A ADVANCED CELL DIAGNOSTICS RNAscope 2 5 HD Reagent Kit RED Each RNAscope 2 5 HD Reagent Kit RED Cat No 322350 provides enough reagents to stain 20 tissue 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 components Pretreatment Reagents Target Retrieval Reagents Wash Buffer Reagents and Detection Reagents IMPORTANT Directions on using the Pretreatment Reagents are inc
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