RNAscope Troubleshooting Tips_July 14 2015
Contents
1. i RNASCOPE TROUBLESHOOT a G b TOPICS RNAscope Recommended Workflow Tips for RNAscope Manual and Automation Assays Troubleshooting Staining Patterns Q amp A RNASCOPE 9 WORKFLOW PERMEABILIZE HYBRIDIZE AMPLIFY VISUALIZE QUANTIFY gt to target RNA gt gt gt cells or tissue signal with morphology single cell expression TISSUE CELLS PreAMP AMP LABEL PROBE SINGLE MOLECULE SEEN AS PUNETATE DOT BREAKTHROUGH PLATFORM SIGNAL SINGLE ANY Amplification Background Molecule Detection Genome Gene Probe Design Suppression in Single Cells or Tissue UNIQUE Chromogenic Diaminobenzene Channel 1 C1 Probes VS LS Probes Chromogenic Fast Red ALP Channel 1 C1 Probes VS LS Probes Chromogenic HRP Green Fast Hed ALP Channel 1 2 C1 C2 Probes Fluorescent FITC Cy3 Cy5 Channel 1 2 3 C1 C2 C3 Probes 2 0 HD AMPLIFICATION SCHEMATIC Amplification Amp6 Brown Enzyme HRP Amplification Amp6 Red Enzyme ALP steps Label probe Substrate DAB steps Label probe Substrate FastRed kjo 3 Je Pre Amp AMP1 Pre Amp AMP1 22 1 2 0 HD BROWN 2 0 HD RED Brown dot Red dot AP Fast Hed TIP Do not interchange reagents within Brown Red assays or across similar 2 0 H2. 3 EASY STEPS b MANUAL ASSAY STEP 1 START WITH STANDARD CONDITIONS STEP 2 STEP 3 ADJUST PRETREATMENT 2 BOILING TIME ADJUST PRETREATMENT 3 4 PROTEASE TIME 10 15 20 25 30 15 20 25 30 PRETREAT 2 BOILING TIME MIN PRETREAT 3 PROTEASE TREATMENT TIP For cultured cells protease is diluted 1 15 in 1X PBS For fresh frozen samples only protease pretreatment is required and is performed at room temperature and NOT at 40 C dd OPTIMIZE YOUR SAMPLE WITH THESE STEPS AUTOMATED ASSAYS Increase EH2 time in increments of 5 mins and protease in increments of 10 mins Increase Pretreat 2 3 and or CC time ene ee VE Decrease Pretreat 2 3 and or CC time morphology and reduces background TIP Refer to the User Manuals for automation assay workflow and pretreatment optimization guideline b EXAMPLE OF SUCCESFUL RNASCOPE RESULTS Target probe WU je RNAscope 2 0 HD Red Assav Human breast cancer tissue Visit http www acdbio com technical support downloads rnascope ish guide troubleshooting for more information PIB Atto 550 RNAscope Multiplex Fluorescent Assay Amp 4 ALT A TIP Use different AMP4 ALT reagents A B C for alternative color combinations Human Hela Cell Line STAINING PATTERNS CHROMOGENIC MANUAL ASSAYS 38 TROUBLESHOOTING NO STAINING OBSERVED PROBABLE CAUSE SUGGESTED ACTION Subopti
3. Pretreat 1 1 5 hours we Pretreat Protease Pretreat 3 or 4 a D Label probe hybridization 1 5 hours Stain and detect Detection Download manuals http www acdbio com technical support downloads RNAscOpe user workflow gt RNASCOPE WORKFLOW FLUORESCENT ASSAY Steps Description Pretreatment conditions will vary based sample type Download manuals http www acdbio com technical support downloads Time to Completion Dg ONE DAY OR TWO DAY ASSAY ONE DAY ASSAY TWO DAY ASSAY Sample preparation Sample preparation DAY 1 oample pretreatment D RNAscope assay sample pretreatment RNAscope assay Review the User Manuals PART 1 and PART 2 for optional stopping points Refer to the User Manuals for Automation assay workflow TIPS FOR RNASCOPE MANUAL ASSAYS e 12 FOR MANUAL ASSAYS Follow Review Alwavs use Review that protocols sample control you are exactly as pretreatment probes using all described in recommenda and slides required the user tions materials manuals PROTOCOLS SAMPLE USE CONTROLS THE CHECKLIST PRETREATMENT Visit http www acdbio com technical support downloads rnascope ish guide troubleshooting for more information on tips for manual assays Da REVIEW THE CHECKLIST Positive and Negative
4. or tapping technique to remove residual reagent Do not let slides dry out Make sure the hydrophobic barrier remains intact Do not alter the protocol in any way SIK Warm probes and wash buffer at 40 C due to precipitation Maintain adequate humidity in the Humidity Control Chamber Fresh reagents ethanol xylene 10 NBF QR Visit http www acdbio com technical support downloads rnascope ish guide troubleshooting for more information on tips for automation assavs 4 POINTERS FOR RNASCOPE 2 0 RED ASSAY E e n v e Use Ecomount as the mounting medium dehydrate sample with alcohol to avoid a diffused signal Samples should be dried in a 60 degree oven for 15 minutes before mounting POINTERS FOR RNASCOPE 2 PLEX CHROMOGENIC ASSAY Probes C2 C1 1 50 Amp 4B Amp 4A 1 50 Red B Hed A 1 60 Green B Green A 1 50 TIP By default C1 probes are 1X concentration while C2 probes are 50X To make 2 plex probe mixture at 1X concentration mix C2 probes 1 50 with C1 probes To view C2 probes only use the blank probe C1 as a diluent and mix at a 1 50 dilution Use Ecomount or PERTEX as the mounting medium dehydrate sample with alcohol to avoid a diffused signal Samples should be dried in a 60 degree oven for 15 minutes before mounting POINTERS FOR RNASCOPE MUL
5. the contents in the containers as this will form bubbles LS Amp 1 LS Amp 3 10X LS Wash Buffer and all target probes require warming up at 40 for 30 mins LS Brown and LS Red assays utilize Leica Biosystems Bond Polymer Refine Detection and Bond Polymer Refine Red Detection kits respectively Do not alter the staining protocol in any way Visit http www acdbio com technical support downloads rnascope ish guide troubleshooting for more information on tips for LEICA BOND RX automation assays USING CONTROLS e 27 IMAGE ANALYSIS RNASCOPE SCORING GUIDELINE a 9 4 10 dots cell QUALIFY YOUR SAMPLES USING CONTROLS a 9 Control 3 6 slides QC check Sample QC check e g Hela gt 2 PPIB 2 PASS FI DapB 1 DapB 41 your Negative control Negative control target probes probe e g DapB probe e g DapB FAIL technique Positive control Positive control Check RNA quality with probe e g PPIB probe e g PPIB new samples Technique check Sample RNA quality check ASSAY OPUMIZANON ED TIP Always start with standard conditions Technique check Sample RNA quality check TIP Refer to the Troubleshooting Guide OPTIMIZE YOUR ASSAY SAMPLE QUALIFICATION QC STEP 1 STAIN SLIDES WITH RNASCOPE ASSAY TEST INVALID Run your samples with Hela or 3T3 Control sl
6. D Assays By default 2 0 HD assays require C1 probes that ready to use no further dilution is required b 2 PLEX AMPLIFICATION SCHEMATIC Amplification Amp6 Brown Enzyme HRP Amplification Amp6 Red Enzyme ALP steps Substrate Green steps Substrate FastRed 9 e e e e e e Pre Amp AMP1 Pre Amp AMP1 2201 ZZ C2 mRNA 1 mRNA Green dot Red dot HRP DAB AP Fast Red TIP By default C1 probes are 1X concentration while C2 probes are 50X To make 2 plex probe mixture at 1X concentration mix C2 probes 1 50 with C1 probes Q To view C2 probes only use the blank probe C1 as a diluent and mix at a 1 50 dilution p RNASCOPE 9MULTIPLEX FLUORESCENT SCHEMATIC Amp3 C1 Amp3 C2 Alexa 488 7 4 Amp3 C3 Atto 550 Atto 647 Amp4 Alt A Amp4 Alt A TEAM 9 Ext Amp1 C1 Amp1 C2 Amp1 C3 MNAI mRNA mRNA Alexa 488 Atto 550 Atto 647 Ex Em 495nm 520nm 555nm 575nm 645nm 670m TIP By default C1 probes are 1X concentration while C2 and C3 probes are 50X To make 3 plex probe mixture at 1X concentration mix C2 and C3 probes 1 50 with C1 probe If C2 and C3 are all at 50X concentration use the blank probe C1 as a diluent and mix at a 1 50 dilution D d RNASCOPE WORKFLOW CHROMOGENIC ASSAY RNAscope user workflow Steps Description Time to Completion Deparaffinization we H O block
7. Normal EN Testis Normal Mild RED BESE Kidney Normal Extended undi o Colon Tumor standard ull i ol Lymph node Tumor Nevus TMA Benign Standard prende TE Tonsil Normal Mild Stomach TMA Tumor pm m EREBE Cervical Cancer Standard EE Ta 7 min 15 min 30 min For information about species or tissue type not listed here contact support at support acdbio com Refer to the user manual for tissue specific pretreatment guidelines Guidelines for Optimal Tissue Pretreatment Test representative samples with positive and negative control probes Controls should be Positive uniform signal negative blank Fix sample in FRESH 109 NBF for 16 32 HOURS at ROOM TEMPERATURE NOTE Do not fix at 4 DO NOT fix for lt 16 hrs gt 32 hrs Refer to Table 1 for under over fixed tissue pretreatment guidelines Vary PRETREATMENT 2 amp 3 and or CELL CONDITION Boiling time TIME based on your tissue type see Table 2 This tissue optimization guide is recommended for Ventana Discovery TROUBLESHOOTING TIPS MULTIPLEX FLUORESCENT ASSAY 53 TROUBLES 40 protease pretreatment 4 Over digested RT protease pretreatment 4 Optimal Fresh Frozen Mouse Kidney Fresh Frozen Mouse Brain Pretreatment temperature has a great effect on the success o
8. TIPLEX FLUORESCENT ASSAY COLOR MODULE OPTIONS AMP 4 AIt A GREEN Alexa 488 ORANGE Atto 550 FAR RED Atto 647 Amp 4 Alt B ORANGE Atto 550 GREEN Alexa 488 FAR RED Atto 647 Amp 4 Alt C ORANGE Atto 550 FAR RED Atto 647 GREEN Alexa 488 TIP By default C1 probes are 1X concentration while C2 and C3 probes are 50X To make 3 plex probe mixture at 1X concentration mix C2 and C3 probes 1 50 with C1 probe If C2 and C3 are all at 50X concentration use the blank probe C1 as a diluent and mix at a 1 50 dilution TIPS FOR RNASCOPE AUTOMATED ASSAYS e 23 TIPS FOR AUTOMATION ASSAYS SYSTEMS Visit http www acdbio com technical support downloads rnascope ish guide troubleshooting for more information on tips for automation assays POINTERS FOR RNASCOPE ON IHE VENTANA DISCOVERY XT OR ULTRA ASSAY Check Instrument Maintenance Pertorminstrument maintenanoe Perform decontamination protocol every three months prevents microbial growth C Use appropriate buffers for RNAscope assay remove or purge before a run Optimize Software Settings Uncheck the Slide Cleaning option Use appropriate hybridization temperature different for XT versus ULTRA TIP This is a cleaning step in Ventana Equipment may cause the slides to dry out Refer to User Manual for details iN POINTERS FOR LEICA BOND RX Do not shake
9. ____ _ CAUSE Tissue detaches from slides Wrong slides used Suboptimal tissue preparation Conditions used for manual assays TROUBLESHOOTING SAMPLE PREPARATION XENOGRAFT FFPE TISSUE hemm Increased baking bv 1 hour SUGGESTED ACTION Use only SuperFrost Plus slides tissue samples according to ACD recommended procedures Bake slides for a longer time up to overnight Heduce boiling time 4 TROUBLESHOOTING SUB OPTIMAL FIXATION CONDITIONS 24 hours fixation Optimal 3 weeks fixation Over fixe naptophysin sample FFPE brain sample Assay RNAscope 2 0 HD Brown TIP Sample fixation has a great effect on the success of your assay Solution Increase pretreatment for better target accessibility TROUBLESHOOTING OTHER ISSUES ISSUE o PROBABLE CAUSE SUGGESTED ACTION Unknown tissue eSample provider clinical Follow the appropriate Tissue Specimen preparation method site vendor did not provide detailed Preparation and Assay Optimization instructions Guidelines Technotes eStart with standard conditions Optimize your assay Diffused Signal RED Sample not completely dried eDrv sample as recommended prolonged Alcohol used to dehydrate sample drying i e overnight may be required e 00 much Ecomount mounting Do not dehydrate samples dry at 60 C medium used 15 min Use Ecomount sparingly and as recommended Applies to all s
10. amples used with RNAscope 4 VERUS TORALENUE UNDER FIXATION Sample Flash Frozen followed by FFPE sample preparation fixation Rat intestines Assay RNAscope 2 0 HD Brown Issue Weak staining destroyed morphology FFPE sample is under fixed Positive control Rn PPIB Optimization Fixation according to recommended guidelines for FFPE samples Result Strong staining for positive control PPIB intact morphology TER gt e Positive control Hn PPIB i fo Refer to the t Guide http www acdbio com technical support downloads rnascope ish guide troubleshooting TROUBLESHOOTING GREEN SIGNAL FADING Sample FFPE human tonsil sample Assay RNAscope 2 plex assay G 8 Issue Green signal faded gt NUS Mon ek we Lo RE B NOS N SN es Mete Probable cause 2 NNI LABOR ELE ta Hematoxylin or associated low pH eBluing with ammonia water Solution Use hematoxylin briefly as recommended 30 secs Use tap water instead of ammonia water e 2555 eo Ps d iS KA Hs Kapp Lambda D RNASCOPE PRETREATMENT GUIDE MANUAL ASSAYS Tissue Pretreatment Guidelines Table 2 Tissue Pretreatment Table Follow the recommended pretreatment conditions ane cee a ee ee mma ee Any new or previously untested FFPE tissues IR Samples prepared suboptimally Embryo Normal Bra
11. control probes Hot Plate for pretreatment target retrieval step ouperftrost plus slides HybEZ Hybridization system Run RNAscope control slides Ecomount for 2 0 HD Red amp 2 plex chromogenic assay Fresh reagents ethanol xylene 10 NBF KIKI KI KI TIP Visit www acdbio com go for more information on getting started Checklist is available on the website and in the manual HOI PLATE Hotplate for retrieval boiling S TIP When using a hot plate for pre treatment step p pay close attention to the TIME and boiling TEMPERATURE 4 RNASCOPE 9 REAGENT KIT CONTENTS NEW OLD Contents of the reagent kit 1 Pretreatment reagents 2 detection kit 3 Wash buffer TIP Warm probes at 40 C for 10 minutes before use TIP Warm 50x wash buffer at 40 C for 20 minutes if you notice a precipitation b HYBEZ HYBHIDIZATION OVEN 7 HyBEZ hybdrization system HybEZ oven is required as it provides both temperature and humidity control necessary to obtain optimal RNAscope results p b ACCESSORIES FOR WASHING STEPS Tissue Tek washing tray EZ Batch for slide processing TIP ACD EZ Batch slide processing tray is easy and convenient for loading multiple slides for hybridization and washing steps f 4 FOLLOW WORKFLOW GUIDELINES MANUAL Apply all amplification steps in the right order 0 F Use flicking
12. cope technical notes and you to get the best product frequently asked workflow videos on our publication lists gene MSD5 result from questions or add one of Video page lists and download dayl your own product brochures Go gt b QUESTIONS PLEASE COMPLETE THE WEBINAR SURVEY WE VALUE YOUR FEEDBACK ACD Advanced Diagnostics 2013 Advanced Cell Diagnostics Inc Confidential and Proprietary
13. f your assay Solution Perform pretreatment at RT to avoid over digestion of your sample Nzu10d 041002 SAIISOd xejd z TROUBLESHOOTING SAMPLE DIGESTION 2 plex Mouse Positive Control Probe Mm POLR2A Fresh Frozen Mouse Brain Experiment condition 10 NBF 15 min Fixation Pretreatment 4 TIP Sample thickness can affect signal in your samples Solution Use recommended sample thickness 10 20um TROUBLE SHOOTING AUTOFLUORESCENCE Mouse FFPE Kidney Mouse FFPE Intestine Mouse FFPE Colon Mouse FFPE Brain TIP FFPE sample have inherent autofluorescence Solution Use appropriate background correction software to reduce autofluorescence MULTIPLEX FLUORESCENT ASSAY 101 PROBLEMS AND SOLUTIONS SOURCE mm PROBLEM SOLUTION Microscopy No weak signal Wrong filter setting longer Nonspecific signal emission cut off 2 Wrong exposure 3 Inappropriate imaging enhancing with software Sample No weak signal 1 Compromised RNA quality 2 Sample preparation high autofluorescence background on the sample Use correct filter settings Do not use using autoexposure at first verify signal with naked eye Use known image enhancing software e g Nuance Use new sample with good RNA quality Follow the pretreatment guideline recommended Always perform assay with 3 plex positive control and 3plex negative probes to assess RNA quality Always check signal with naked eye under objective lens firs
14. ides Retest with fresh slides s Positive control probe PPIB POLR2A UBC s Negative dapB probe FAIL QC CHECK Hela Control spec POLRZA PPIB 2 gt 3 dapB lt 1 TEST FAIL TEST PASS FAIL SAMPLE CHECK SAMPLE FAIL 9 OPTIMIZATION Your sample QC spec POLR2A PPIB 2 2 Vary pretreat 2 amp or 3 UBC z3 dapB 1 PASS RETEST RUN YOUR TARGET MARKERS SAMPLE PASS Repeat Steps 1 2 amp 3 with optimized RNAscope assay SAMPLE FAIL Contact ACD support amp acdbio com OPTIMIZE YOUR ASSAY gt WHY OPTIMIZE YOUR RNASCOPE ASSAYS Under fixed when using the following conditions 49 24 hours 4 C 4 PFA lt 24 hours RT 10 NBF 24 hours 4 C 4 PFA 24 hours 4 C Over fixed when using the following conditions 10 NBF gt 48 hours RT 1096 NBF gt 48 hours 4 C Special sample types Cultured cells pellet FACTORS AFFECTING RNASCOPE ASSAY PERFORMANCE ES en RNA is degraded Hybridization conditions not optimal oamples drying during assay opecial tissues sensitive to pretreatment THE SOLUTIONS Acquire new samples and assess RNA quality Use the HybEZ hybridization oven only Use Immedge pen and add adequate reagents to avoid drying 4 SE otart with standard pretreatment then optimize conditions accordingly NBF Neutral Buffered Formalin OPTIMIZE YOUR SAMPLE IN
15. in Homa Guidelines for Optimal Tissue Pretreatment upeen Homa Testrepresentative samples with positive and negative control probes Controls should be ye Retin Normal Positive uniform signal negative blank L Fix saniple in FRESH 10 NBF for 16 32 Klaney Horma NOTE Do not fix at FC DO fix for B lt 16 hrs gt 32 hrs Refer to Table 1 for Coon Tumor yer d tissue pretreatment colon under over fixed tissue guidelines C Vary PRETREAT 2 and or PRETREAT ae based on your tissue type see Table 2 Lung Normal NOTE Certain Xenografts and Cell Prostate Tumor Pellets mild ez require m min PRETREAT 3 for Table 1 Tissue Pretreatment Guidelines Tonal Mormal 15 min i13 man Pretrect 15 min 30 min 30 min Cervical Cancer For information about species or tissue type not listed here contact support at support acdhio com TIP Refer to the user manual for tissue specific pretreatment guidelines RNASCOPE PRETREATMENT GUIDE VENTANA DISCOVERY ULTRA SYSTEMS Tissue Pretreatment Guidelines AED Table 2 Tissue Pretreatment Table Follow the recommended pretreatment conditions based on your tissue type for gt Any previously untested FFPE tissues Samples prepared suboptimally je ee Kidney Normal Standard pro qe ER Melanoma Standard ee p __________ Breast TMA
16. mal fixation Prepare fix samples according to ACD Over fixation recommendation Under fixation Optimize pretreatment conditions Hybridization temperature not optimal Use HybEZ when performing RNAscope HybEZ temperature should be at 40 Reagents used in the wrong sequence Apply reagents in the correct order Gene of interest not expressed Check positive control for technical accuracy of the assay 24 TROUBLESHOOTING SAMPLE DIGESTION OPTIMAL DIGESTION XENOGRAFT TISSUE X Hs PPIB 8 min Pretreat 2 30 min Pretreat 3 ao Conditions used for manual assays TROUBLESHOOTING SAMPLE DIGESTION ON 8 min Pretreat 2 15 min Pretreat 3 MC m Hs PPIB Assay HNAscope 2 0 HD HED Issue Strong hematoxylin under pretreatment weak PPIB Solution Increase pretreatment 44 Conditions used for manual assays TROUBLESHOOTING SAMPLE DIGESTION OVER MIREN _XENOGRAFT TISSUE a d m LHE T Li 15 min Pretreat 2 30 min Pretreat Hs PPIB Assay HNAscope 2 0 HD HED Issue Nuclear background over pretreatment Solution Decrease pretreatment 45 Conditions used for manual assays TROUBLESHOOTING BACKGROUND STAINING HIGH BACKGROUND _ KIDNEY FFPE TISSUE DapB ni T m Za 15 min Pretreat 2 30 Pretreat 7 Pretreat 2 30 min Pretreat 3 Assay HNAscope 2 0 HD BROWN Issue High backgr
17. nd no signal sample detachment Optimizing with Pretreatment 2 and 3 optimization MANUAL Adjusting ER2 protease time and hyb temperature changes LEICA Offline online pretreatment optimization CC and pretreat 2 3 VENTANA VISIT THE SUPPORT PAGE TO LEARN MORE ie Provider the most Contact Us Quote Builder sensitive and specific Advanced Cell Diagnostics in situ hybridization technology Applications Products Technical Support Overview Webinars Download Manuals User Manual Selection Guide Manuals amp MSDS Positive amp Negative Control Images Tissue Requirements amp Troubleshooting Tips Getting Started Troubleshooting Guide Act Frequently Asked Questions FAQs Online Training Videos Product Literature Brochures Probe Lists CHOOSE DOWNLOAD IT WORKS GA ER ASSAY MANUALS Application Reviews Researcher in the spotlight mytechnical support support overview scientific Posters 2 Visit www acdbio com technical support support o verview CONTACT ACD SUPPORT gt Support via email support acdbio com gt Support 1 877 376 3636 option gt Time 8 00am 6 00pm PST gt Support Resources available on website www acdbio com Wess Cc e Product Started Literature Download manuals simple tips amp tricks for Browse through our View our product and Find RNAs
18. ound over pretreatment Optimization Decrease pretreatment 2 boiling conditions Result Clean background a Conditions used for manual assays 44 TROUBLESHOOTING BACKGROUND STAINING NUCLEAR HAZY BACKGROUND HUMAN TONSIL FFPE TISSUE DapB 15 min ER2 30 min Protease 20 min ER2 30 min Protease Assay RNAscope LS BROWN LEICA BOND RX Issue Nuclear hazy background under pretreatment Optimization Increase ER2 time in increments of 5 mins and protease in increments of 10 mins Result Clean background o Conditions LEICA BOND RX automated assays gt TROUBLESHOOTING ASSAY WORKFLOW HIGH BACKGROUND DRYING FFPE HELA PELLET T aa DapB 15 min Pretreat 2 30 min Pretreat BACKGROUND TYPE PROBABLE CAUSE SUGGESTED ACTION Cytoplasmic and nuclear Samples drying between amplification steps Completely cover tissue when applying reagents Process slides one at a time to prevent drying Ensure HybEZ Oven is at the appropriate temperature Use the Immedge hydrophobic barrier pen incomplete paraffin removal eUse fresh unused EtOH and Xylene and agitate slides during eSuboptimal tissue preparation incubation steps tissue samples according to ACD recommended rocedures 45 Conditions used for manual assays Extracellular 46 SAMPLE FALLING OFF om 25 Shere DapB Standard baking protocol B ST _______
19. t MULTIPLEX FLUORESCENT ASSAY 101 TIPS AND TRICKS Be aware of the suggested filter settings for your microscope Use the suggested pretreatment condition Use the sample preparation protocol PART 1 for your samples for optimal results Always run a 3 plex positive control and negative control to assess RNA quality and to verify microscope setting are appropriate Always evaluate the results by eye first before capturing images FREQUENTLY ASKED QUESTIONS e 29 FREQUENTLY ASKED QUESTIONS RNAscope assay compatibility with different tissues RNAscope manual assay can be used with FFPE fresh frozen fixed frozen and cultured cells RNAscope automated assays are primarily supported with the FFPE tissue Please refer to the User Manual Selection Guide http www acdbio com technical support downloads Key differences between RNAscope ISH assay and IHC for FFPE samples No cooling Is required during Epitope retrieval users should directly put the slides in water at room temperature dehydrate and proceed to Pretreatment 3 step as per the manual Part 1 60 TIP Visit www acdbio com support for additional FAQs od b SUMMARY RNAscope recommended workflow for Manual assays Automated assays NO Tips for RNAscope amp manual and automation assays Check instrument maintenance Optimize software settings Optimize your assay 3 Troubleshooting staining patterns High backgrou
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