- Advanced Cell Diagnostics
Contents
1. 14 Prepare regens oec eie ee tir Reb e Maa REM MER MEE 14 Prepare the instrument 14 Register the WES IIS sei descciaceesecacebessoadebess oudetassvetevascvarevacsveisvesustsuacevaer 15 Prepare instrument 16 Set up the 16 Create a prestaining protocol 2 tici ttu 16 Create a staining protocol si eie cote eto dr o te tele rte eite 22 Register ERN 26 Set up a study Mm 28 Run the RNAscope 30 Materials required sss 30 Load the Reagent Soon E E QU PU IU EE 30 Stark he rM oe 31 Complete the run 31 RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red Mount desti ee ee baee tied etd etn 31 Chapter 5 Evaluate the 33 Scoring guidelines NIETO 33 Quantitative Image Analysis eese eene rnnt nnns 33 Control excniple ci2000 candela eee 34 NNI a a ee ae ee ne ne ene nee ere 34 Appendix A Safety ccsccssccssessssscessccssessesseessessceesessesaeenseeaees 35 Chemical To I ERIT IO 35 Biological hazard safely 1 bean bn 35 Docum2. 21 22 4 Highlight the No Reagent step Change the incubation time to 120 and temperature to 40 5 Select Save Create a staining protocol 1 In the Protocol setup screen select Staining under the Protocol group menu BOND RX Protocol screen File Window Item ID Configuration Maintenance IHC Protocol B IHC staining Bond Int rot IHC Protocol F IHC staining Bond Polymer Refine IHC protocol MHC Protocol FRX37M IHC staining IHC Protocol F with marker step at 37C IHCProtecol FRX40M IHC staining IHC Protocol with marker step at 40C Protocol J IHC staining Bond Polymer Refine Red IHC protocol AHC Protocol JRX37M IHC staining IHC Protocol J with marker step at 37C AHC Protocol JRX40M IHC staining IHC Protocol J with marker step at 40C IHC Protocol K IHC staining ChromoPlex 1 Dual IHC protocol FISH A RUO Protocol ISH detection Bond RUO FISH protocol ISH Protocol Bond Polymer Refine RNAISH protocol Leic 14 01 2013 IBHProtocolB on Bond Polymer Refine DNAISH protocol 7 I amp IX amp IXIXI amp XIX T ISH detection Affymetrix ViewRNA eZ Assay Alternate 2 I8H detection Affymetrix ViewRNA eZ Assay Alternate 3 aa Protocol group Protoc l type Protocol origin Preferred status EA Leica Microsystems 2 Highlight the ViewRNA 1 FFPE protocol Select Copy 3 Change the name to ACD ISH Red Protoc
3. Antigen Retrieval 1 Under the Protocol type menu select Heat pretreatment HIER 20 min with ER2 20 min HeatRetrieval using ER2 HIER 25 min with 97 Heatpretreatment 25 HeatRetrieval using ERT X 1 14 2013 HIER 30 min with ER1 Heatpretreatment 30min HeatRetrieval using ER1 Lei 1 14 2013 HIER 30 minwithER2 Heatpretreatment 30 min HeatRetrieval using ER2 1 14 2013 HIER 40 min with ERT Heatpretreatment 40 HeatRetrieval using ER1 ica 14 14 2013 HIER 4 minwithER2 Heatpretrestment 40min HeatRetrieval using ER2 171412013 HIER 5 min with ERT Heatpretreatment 5 min HeatRetrieval using ERT i 1n4 013 Protocol group Protocol type Staining statu Protocol origin Preferred status Prestaining T Heat pretreatment RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 18 2 Highlight the HIER 10 min with 2 protocol Select Copy Protocol origir 3 Rename the protocol e g ACD 15 95 C Change the Abbreviated name e g ER2 15 and the Description e g ACD 15 min ER2 95 FA BOND Praienl creen New protocol properties fa T ACD 15min ER2 995 C Protocoltype Abbreviated name ER2 15 RX 1 ACD 15min ER2 amp 85 Step details Reagent Incubation time min Wash Temperature C Show wash steps Preferred 31 RNAscope LS Assay for Leica Biosystems
4. Bond Wash Solution Wash O MIN Ambient 15 Bond Wash Solution Wash O MIN Ambient 16 Bond Wash Solution Wash O MIN Ambient 17 ACD Amp 2 Reagent 1 MIN 42 18 ACD Amp 2 Reagent 15 MIN 42 C RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 23 24 Step No Reagent Step Type Incubation Time Temperature 19 Bond Wash Solution Wash O MIN Ambient 20 Bond Wash Solution Wash O MIN Ambient 21 Bond Wash Solution Wash O MIN Ambient 22 Bond Wash Solution Wash 1 MIN Ambient 23 Bond Wash Solution Wash 1 MIN Ambient 24 Bond Wash Solution Wash 1 MIN Ambient 25 Bond Wash Solution Wash 1 MIN Ambient 26 Bond Wash Solution Wash 1 MIN Ambient 27 ACD Amp 3 Reagent 1 MIN 42 28 ACD Amp 3 Reagent 30 MIN 42 29 Bond Wash Solution Wash O MIN Ambient 30 Bond Wash Solution Wash O MIN Ambient 31 Bond Wash Solution Wash O MIN Ambient 32 Bond Wash Solution Wash 3 MIN Ambient 33 Bond Wash Solution Wash 3 MIN Ambient 34 Bond Wash Solution Wash 1 MIN Ambient 35 Bond Wash Solution Wash 1 MIN Ambient 36 Bond Wash Solution Wash 1 MIN Ambient 37 ACD Amp 4 Reagent 1 MIN 42 38 ACD Amp 4 Reagent 15 MIN 42 39 Bond Wash Solution Wash O MIN Ambient 40 Bond Wash Solution Wash O MIN Ambient 41 Bond Wash Solution Wash O MIN Ambient 42 Bond Wash Solution Wash 1 MIN Ambient 43 Bond Wash Solution Wash 1 MIN Ambien
5. 10 mL x 1 bottle 4 C LS Pretreat 3 Protease 21 mL x 1 bottle 4 C LS Amp 1 21 mL x 1 bottle 4 C LS Amp 2 21 mL x 1 bottle AC LS Amp 3 21 mL x 1 bottle AC LS Amp 4 21 mL x 1 bottle 4 C LS Amp 5 Red 21 mL x 1 bottle AC LS Amp 6 Red 21 mL x 1 bottle AC LS 10X Wash Buffer 5 ml x 1 bottle 4 C IMPORTANT Do not substitute the reagent components of the RNAscope Reagent Kit with those of other RNAscope Reagent Kits even those having the same name Required materials from Leica BOND RX The RNAscope LS Assay requires specific materials and equipment available only from Leica Biosystems Cat No Storage BOND Open Containers 30 mL Op309700 Room temperature 20 25 BOND Universal Covertiles 100 pack 521 2001 Room temperature 20 25 C BOND Epitope Retrieval Solution 1 1L RTU AR9961 2 8 C BOND Epitope Retrieval Solution 2 1L RTU AR9640 2 8 C BOND Dewax Solution 1L RTU AR9222 2 8 C BOND Wash Solution 10X Concentrate 1L AR9590 2 8 C BOND Polymer Refine Red Detection and Hematoxylin DS9390 2 8 C BOND Aspirating Probe Cleaning System 59100 2 8 C BOND Mixing Stations 521 1971 Room temperature 20 25 Do not substitute with any other chromogen kit Equipment Cat No Leica Biosystems BOND RX System automated slide stainer RNAscope LS Assay for Leica Biosystems BOND RX S
6. Manval Red 4 Chapter 4 Fully Automated RNAscope LS Assay IMPORTANT We strongly recommend you run the RNAscope Control Slides Cat No 310045 or Cat No 310023 using the RNAscope LS positive and negative control probes along with your samples in every run Workflow Prepare the materials i Set up the instrument i Run RNAscope Assay 9 hours RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red Prepare the materials Materials can be prepared ahead of time unless otherwise stated Materials required Materials provided by Advanced Cell Diagnostics Materials provided by Leica Biosystems Materials provided by user RNAscope LS Target Probe RNAscope LS Positive Control Probe RNAscope LS Negative Control Probe LS Pretreat 1 15 Pretreat 3 LS Amp 1 LS 2 LS Amp 3 LS Amp 4 LS Amp 5 Red LS Amp 6 Red LS 10X Wash Buffer Stainer Leica Biosystems BOND RX System Bulk Reagents BOND Wash Solution e BOND Dewax Solution e BOND Epitope Retrieval Solution 1 e BOND Epitope Retrieval Solution 2 Reagents e BOND PolymerRefine Red Detection plus Hematoxylin Distilled water Conical tube 50 mL Drying oven Prepare the reagents e Warm up LS Amp for 30 MIN before the run Note Loss of signal will occur if precipitates do not dissolve LS Amp 3 LS 10X Wash Buffer and all LS target
7. Re reen rie window Item iD Configuration Mainterenc 3 Add reagent a ancillary E ETT ACD Available Compatible bulks i BWash DI Preterred O Hazardous Reagent type Supplier Preferred status Enter ACD Amp 1 in the Abbreviated name text box Select Ancillary in the Type drop down menu Enter ACD in the Supplier text box Select Save Repeat steps 2 7 for Amp 2 Amp 6 Red and 1X LS Wash Buffer RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 15 16 ACD Note not add reagents for LS Pretreat 1 and LS Pretreat 3 They will be directly scanned and registered as Open 0 Haz and Enzyme 1 respectively See Prepare instrument reagents Prepare instrument reagents Fill the Leica BOND RX containers with the appropriate reagents from the RNAscope LS Reagent Kit according to the following table Reagents Container Name LS Pretreat 1 Open 0 Haz LS Pretreat 3 Enzyme 1 LS Amp 1 ACD Amp 1 15 Amp 2 ACD Amp 2 15 Amp 3 ACD Amp 3 LS Amp 4 ACD Amp 4 LS Amp 5 Red ACD Amp 5 Red LS Amp 6 Red ACD Amp 6 Red LS 1X Wash Buffer ACD Wash Buffer LS Target Probe Variable Note Leica BOND Red and Hematoxylin come in prefilled Leica BOND RX containers IMPORTANT Do not introduce bubbles into the solutions by shaking the containers To mix
8. Temperature Brain and spinal cord 15 MIN 40 C Breast cancer 15 MIN 40 C Cell pellet 15 MIN 40 Colon 15 MIN 40 15 40 Head and neck cancer 15 MIN 40 Heart 15 MIN 40 Kidney 15 MIN 40 25 40 Lung 15 MIN 40 Lymphoma 15 MIN 40 Placenta 15 MIN 40 Prostate 15 MIN 40 Skin 15 MIN 40 Stomach 15 MIN 40 Thymus 15 MIN 40 Tonsil 15 MIN 40 Xenograft 15 MIN 40 RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red Select Save Probe Hybridization Y Highlight the Open 0 Haz step Change the incubation time to 10 MIN 1 In the Protocol setup screen select ISH Hybridization under the Protocol type menu BOND RX Protocol screen File Window ItemID Configuration Maintenance Help 1 Protocol setup Protocol group Pr Highlight the ISH Hybridization 2Hr protocol Select Copy Prestaining v ISH hybridization YM Description to ACD 2 Hour Hybridization FA BOND Protocol screen New protocol properties ACD 2 Hour Hybridization Protocol type Abbreviated name Hyb 2hr V ACD 2 Hour Hybridization Er IM Step details Incubation time miny Temperature C RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 3 Change the Name to ACD 2 Hour Hybridization the Abbreviated Preferred
9. in the Name and Abbreviated name text boxes Select Probe in the Type drop down menu Enter ACD in the Supplier text box Check RNA for Probe Type Select ACD ISH Red Protocol as the Default staining protocol Select ACD 15min 95 as the Default HIER protocol Select ACD 15min Protease as the Default enzyme protocol leave the Default denaturation protocol blank 10 Select ACD 2 Hour Hybridization as the Default hybridization protocol 11 Select Save 12 Repeat steps 2 11 for each additional probe RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 27 28 ACD Set up a study b je I FA BOND Sys odule 1 To build a study select the Slide setup icon at the top of the screen System status Lans Lami Ld Lam Lene Lom Lem omnl aani Led Lem md d Ld Lnd Lem Lem end a Lem Lem Lom Lm Lam m m m Ln StuayiD Test Run 1 7 Study comments Sey study Ne UNES Researchers ist 7 Dispense volume Select 150 yl for Dispense volume Select 150 yl for Preparation protocol Select OK RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 6 Select Add slide to assign a protocol to each slide 7 Enter the name of the tissue under the Comments field Then select ISH Select the marker Target Probe BOND RX Sli Study name Study comments d so CE C
10. on the instrument in 9 10 hours Starting with properly prepared samples sections are first pretreated and then probes are hybridized to target RNA The signal is amplified using multiple steps followed by hybridization to alkaline phosphatase APHabeled probes and detection using Fast Red Each single RNA transcript appears as a distinct dot of chromogen precipitate visible using a common microscope RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red Figure 1 Procedure overview o ZZ Target RNA Specific Oligo Probes PreAMP AMP Label Probe 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 load slides onto the in binding of AP labeled probes Add Fast standard bright instrument Pretreat tissue Red substrate to detect target RNA field microscope to allow access to target RNA Kit contents and storage The RNAscope LS Assay requires the RNAscope LS Probes and the RNAscope LS Reagents available from Advanced Cell Diagnostics RNAscope LS Probes The RNAscope LS Probes consist of the userspecified Target Probe and the Positive and Negative Control Probes Visit www acdbio com products target probes search product to find a gene spe
11. reagents gently invert the containers several times If bubbles are present leave the containers out at room temperature until the bubbles dissipate Set up the instrument Create a prestaining protocol 1 Open the instrument software version BDZ 6 0 or higher and click on the Protocol setup icon as shown DI BWash Alcohol BikWast BikWast RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red AD 2 Select Prestaining under the Protocol group menu located in the bottom left corner of the screen to access the Enzyme Pretreatment Heat pretreatment and ISH Hybridization protocols 10 min ig ER2 nt 20min HeatRetrieval using ERT i 1114 2013 HIER 2 minwithER2 Heatpretreatment 20 min HeatRetrieval using ER2 Lei 1 14 2013 HIER25 minwith ER1 87 Heatpretreatment 25min HeatRetrieval using ERT 1 14 2013 HIER 30minwithER1 Heatpretreatment 30min HeatRetrieval using ERT Lei 1114 2013 30min withER2 Heatpretreatment 0 HeatRetrieval using ER2 i 1142013 HIER 40 min with ERT Heatpretreatrnent 40 min HeatRetrieval using ERT ica 114 2013 HIER 40 min with ER2 Heatpretreatment 40 min HeatRetrieval using 2 114 2013 HIER 5 min with ERT Heatpretreatment 5min HeatRetrieval using ERT 14 2013 sets Protocol type Protocol origin Preferred status Prestaining T Heat pretreatment v Leica Microsystems
12. 1 De ionized Water Wash O MIN Ambient RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red Note The temperature for these steps cannot be changed You may on incubation times ly change the 6 Click Show wash steps to view the washing steps in between each reagent Inset BOND Washes to match the protocol steps shown in the table above 25 26 YN 7 Make sure that Preferred is selected at the bottom right corner of the window Register Probes 8 Select Save l ACD ISH Red Protocol ACD Red sl ACD ISH Red Protocol BONURX Preferred detection system Bond Polymer Refine Red Detection x Step details ACD Amp 1 0 v Incubation time min Wash 19 0 Show wash steps Double staining status 0 Preferred single Fist Second Click the Reagent setup icon to register each probe System Protocol Lcd Lami Lanal Led Led Lam Lans Lammi riri RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 2 Select Add FA BOND RX Reage File Window Add reagent E EUC Inventory Panels 4 Single double stain Probe Type Single D rie Detau enzyme Compatible Preferred O Hazardous Cena Enter the name of the probe
13. BOND RX System User Manval Red Ww 4 Highlight the third Bond ER Solution 2 step Change the temperature to 95 C and incubation lime according to the following table Tissue Type ER2 Incubation Time Temperature Brain and spinal cord 15 MIN 95 C Breast cancer 15 MIN 95 C Cell pellet 15 MIN 95 C Colon 15 MIN 95 C Gl tract 15 MIN 95 C Head and neck cancer 15 MIN 95 Heart 15 MIN 95 Kidney 15 MIN 95 20 95 Lung 15 MIN 95 C Lymphoma 15 MIN 95 C Placenta 15 MIN 95 C Prostate 15 MIN 95 C Skin 15 MIN 95 C Stomach 15 MIN 95 C Thymus 15 MIN 95 C Tonsil 15 MIN 95 C Xenograft 15 MIN 95 C 5 Select Save Protease and H 0 Treatment 1 Under the Protocol type menu select Enzyme Pretreatment 2 Highlight the Protease 20 min and fix protocol Select Copy f BOND RX Protocol screen i 7 Bw Protocol setup Coren Enzyme 1 for 10 min Enzyme pretreatment 10 min Enzyme Pretreatment using Enzyme 1 7 44 01 2013 Enzyme 1 for 15 min Enzyme pretreatment 15 min Enzyme Pretreatment using Enzyme 1 Lei 14 01 2013 Enzyme 1 for 5 min Enzyme pretreatment 5 min Enzyme Pretreatmentusing Enzyme 1 T 14 01 2013 Enzyme 2 for 10 min Enzyme pretreatment 10 min Enzyme Pretreatment using Enzyme 2 ii 14 01 2013 Enzyme 2for 15 min Enzyme pretreatment 15 min Enzyme Pretreatment using Enzyme 2 ic 14 01 2013 Enzyme 3for 10 m
14. USER MANUAL Fully Automated RNAscope Assay RNAscope LS Reagent Kit For use with Leica Biosystems BOND RX System RED Document Number 321 127 For Molecular Biology Applications not intended for diagnosis Refer to appropriate regulations Rev Date 20141112 For Molecular Biology Applications not intended for diagnosis Refer to appropriate regulations Trademarks RNAscope is a registered trademark of Advanced Cell Diagnostics Inc BOND is a registered trademark of Leica Biosystems All other trademarks belong to their respective owners Citing RNAscope LS in Publications When describing a procedure for publication using this product please refer to it as the RNAscope LS Assay and cite Wang F Flanagan J Su Wang LC Bui S Nielson A Wu X Vo Ma XJ and Luo Y RNAscope A Novel n Situ RNA Analysis Platform for Formalin Fixed Paraffin Embedded Tissues J Mol Diagnostics 2012 14 22 29 Disclaimers Advanced Cell Diagnostics Inc reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure accuracy Advanced Cell Diagnostics Inc assumes no liability for any errors omissions or for any damages resulting from the use of this information Copyright 2014 Advanced Cell Diagnostics Inc All rights reserv
15. cific Target Probe Visit www acdbio com products target probes controls housekeeping to order appropriate Control Probes Each probe is sufficient for staining 30 standard slides The probes have a shelf life of six months from the shipment date when stored as indicated in the following table Target Probes Cat No Content Quantity Storage RNAscope LS Target Probe Various Probe targeting specific RNA 11 1 bottle 4 C species gene Control Probes v Reagent Cat No Content Quantity Storage RNAscope LS Positive Control Probe Various Probe targeting common 11 mLx1 bottle 4 C species housekeeping gene RNAscope LS Negative Control 312037 Probe targeting bacterial 11 mLx 1 4 C Probe DapB gene dapB RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red RNAscope Reagents The RNAscope LS Reagent Kit RED Cat No 321130 contains all the reagents needed to run the RNAscope Assay on Leica Biosystems BOND RX System except for the RNA specific probes The kits provide enough reagents to stain 60 standard slides The reagents are Ready To Use and have a shelf of six months from the shipment date when stored as indicated in the following table RNAscope LS Reagent Kit RED Cat No 321130 Reagent Quantity Storage LS Pretreat H50
16. cterize by reagents and substra nhalation of chemicals Do not leave chemical containers open Use only with tion or example fume hood analysis if necessary the waste generated by the particular applications es used in your laboratory e Ensure that the wast local state provi e IMPORTANT Radi ncia oacti is stored transferred transported and disposed of according to all and or national regulations ve biohazardous materials may require special handling and disposal limitations may apply Biological hazard safety Q WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regu to p ations Wear appropriate protective equipment which includes but is not limited rotective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped faciliti 5 using the appropriate safety equipment for example physical containment devices Individuals should be trained according to appli cable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regu In the U S atory requirements in the following e U S Department of Health and Human Se
17. ed Contents Chapter 1 Product Information 5 About this ao sace RR 5 Product T 5 5 o 5 Kit contents and storage 6 RNAscope LOIS Se a 6 RNAscope Reagents isses nennen nnne nenne 7 Required materials from Leica BOND 7 Equipment 1 tee ease 7 User supplied moterials 8 Chapter 2 Before You 55 9 Important procedural guidelines sss 9 Chapter 3 Prepare and Pretreat 11 Prepare FFPE sections terne ertet ettet eee tese e dean 11 Materials required ax 22 0 e 2 n 2casacenadecadesadecedenncesesenacedaceadecavecedecacevacecateueee20 11 DUE SNM Se sigte ss stsnsccanseancearoeenasd ceseesescsensacosssesteasdvectansdessinasseneieasens 11 Dehydrate embed and cut the sample sss 11 Chapter 4 Fully Automated RNAscope LS Assay 13 Workflow 13 Prepare 14 Materials
18. entation and support essent 37 Obtaining MSS 37 so serio evite roto Sto pe oto pro bon DIEM 37 Contact information IEEE 37 Limited 37 RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red Chapter 1 Product Information Before using this product read and understand the information in Appendix A Safety on page 35 in this document IMPORTANT We recommend reading the entire user manual before beginning any protocols About this guide This user manual provides guidelines and protocols to use the RNAscope LS Reagent Kit for use with Leica Biosystems BOND RX Research Advanced Staining System RNAscope LS Assays are compatible with a variety of sample types Product description Background The RNAscope LS Assays use a novel and proprietary method of jin sifu hybridization ISH to visualize single RNA molecules per cell in formalin fixed paratfin embedded FFPE tissue mounted on slides The assays are based on Advanced Cell Diagnostic s patented signal amplification and background suppression technology and can detect RNA molecules in archival samples and partially degraded specimens The RNAscope LS Assay allows users to automate the highly sensitive RNAscope Assay using Leica Biosystems BOND RX System Overview The RNAscope LS Assay procedure is illustrated in Figure 1 on page 6 and can be completed
19. her chromogen kits Do not shake the contents in the dispensers as this will form bubbles and may lead to weak or no staining To mix reagents gently invert the dispensers several times If bubbles are present leave the containers out at room temperature until the bubbles dissipate Do not alter the staining protocol in any way except the hematoxylin incubation time The parameters in the staining protocol have been optimized to run the RNAscope assay on the instrument For troubleshooting information please contact technical support at support acdbio com 34 RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red Chemical safety Appendix A Safety preca 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 utions and instructions e 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 e Minimize co ntact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing e Minimize the i adequate ve ntila e Chara
20. in Enzyme pretreatment 10 min Enzyme Pretreatment using Enzyme 3 i 14 01 2013 Enzyme 3for 15 min Enzyme pretreatment 15 min Enzyme Pretreatment using Enzyme 3 ei 14 01 2013 Enzyme 5for25min Enzyme pretreatment 25min Enzyme Pretreatment using Enzyme 5 Leic 14101 2013 d 0 min and fix 9 04 2014 Enzyme pretreatment 20 min enzyme pretreatment for Affymetrix eZ Assay Leica ViewRNA Enzyme 2 20 Enzyme pretreatment 20 min enzyme pretreatment for Affymetrix ViewRNA eZ Assay Leica ViewRNA Enzyme 3 20 Enzyme pretreatment 20 min enzyme pretreatment for Affymetrix ViewRNA eZ Assay Leica Protocol graup Prot Enzyme pretreatment A S T Staining siatus Preferred Siatus RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 20 YN D 3 Rename the protocol e g ACD 15min Protease Change the Abbreviated name e g 15 and the Description e g ACD 15min Protease f BOND RX Pratnenliecreen New protocol properties Ea T ACD 15min Protease Protocoltype ismeo RX1 EET ACD 15min Protease BOND RX Step detail Reagent 5 Incubation time min Wash Temperature C Ambient Show wash steps Preferrect gt 4 Highlight the second Enzyme 1 step Change the temperature to 40 and incubation time according to the following table Tissue Type Enzyme 1 Incubation Time
21. mply load any image select a region of interest define settings and run analysis followed by a quality control review before results are exported RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 33 ACD Control example If the assay is s gt Troubleshooting If you obtain le Figure 2 RNAscope uccesstul the staining should look like the following images Assay detection of PPIB mRNA in mouse lung FFPE tissue EC We M dum ss than satisfactory results troubleshoot your assay by following these simple guidelines If you observe the presence of background staining increase the Epitope Retrieval 2 ER2 in increments of 5 minutes and increase the Enzyme Protease time in increments of 10 minutes Keep the temperatures for each step constant e g 20 min ER2 at 95 C and 25 min Protease at 40 C 25 min ER2 at 95 C and 35 min Protease at 40 Use the above process for overfixed tissues 15 Amp 1 15 Amp 3 LS Wash Buffer and all target probes require warming up prior to running the assay to remove crystals that form during refrigeration Incubate the reagents in an oven or water bath at 40 C for 30 minutes Failure to warm the reagents properly will lead to weak or intermittent staining The RNAscope LS Brown and LS Red assays utilize Leica Biosystems BOND Polymer Refine Detection and Bond Polymer Refine Red Detection kits respectively Do not use any ot
22. ng the scoring guidelines in the next section Scoring guidelines The RNAscope Assay enables a semi quantitative scoring guideline utilizing the estimated number of punctate d An examp staining in copies per Note f cell 05 present within each cell boundary e of how to develop such a guideline for semi quantitative assessment of RNAscope ensily is presented below for a gene with expression level varying between 1 to gt 10 xpression level is higher or lower than this range you may need to scale the criteria according y your gen Categorize staining into five grades 0 1 2 3 and 4 according to the following table Staining Microscope objective scoring score 0 No staining or less than 1 40X magnification 1 1 3 dots cell visible at 20 40X magnification 2 4 10 dots cell No or 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 Quantitative Image Analysis RNAscope Spot Studio Software is designed for pathologists with no prior training in image analysis This intuitive software allows users to get statistical results with complete information of cell count region and number of spots cell Si
23. ning System Refer to the Leica Biosystems BOND RX System Instructions For Use Run the assay on RNAscope Control Slides Cat No 310045 for Human Hela Cell Pellet and Cat No 310023 for Mouse 3T3 Cell Pellet using the RNAscope LS Positive and Negative Control Probes Important procedural guidelines Start with properly fixed and prepared sections Refer to Chapter 3 Prepare and Pretreat Samples on page 11 for preparation of FFPE slides For preparation of other sample types contact support acdbio com Regularly maintain and clean your automated staining instrument 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 all necessary safety procedures Refer to Appendix A Safety on page 35 for more information RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 10 Y D RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 3 Chapter 3 Prepare and Pretreat Samples Formalin tixed paraftin embedded sample preparation and pretreatment are described in th following protocols IMPORTANT We highly recommend following these guidelines We cannot guara
24. ning dish containing distilled water 3 Wash the slides by lifting the slide rack up and down several times Mount the samples 1 Remove the slide rack from the staining dish and dry slides in a 60 dry oven for 15 MIN IMPORTANT Red substrate is alcohol sensitive Do not dehydrate the slides in alcohol 2 Cool the slides for 5 MIN at RT 3 Briefly dip one slide into 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 4 Carefully place 24 mm x 50 mm coverslip over the tissue section Avoid trapping oir bubbles 5 Repeat steps 2 and 3 for each slide Air dry slides for 5 MIN 7 Proceed to Chapter 5 Evaluate the results on page 33 RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 31 32 Y D RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 5 Chapter 5 Evaluate the results Examine tissue sections under a standard bright field microscope at 20 40X magnification e Assess tissue and cell morphology e Assess positive control signal s n rength Positive control signal should be visible as punctate dots within cell nuclei at 20 40 magnification e Assess negative control background One dot to every 10 cells displaying background staining per 2 microscope field is acceptable e Evaluate target probe signal usi
25. ntee assay results with other preparation methods Prepare FFPE sections Materials required 10 neutral buffered formalin NBF 1X PBS Paraffin wax e 100 EtOH e Xylene Microtome e Water bath SuperFrost Plus slides Fix the sample 1 Immediately following dissection fix tissue in 10 NBF for 16 32 HRS at ROOM TEMPERATURE Fixation time will vary depending on tissue type and size CAUTION Handle biological specimens appropriately IMPORTANT Fixation for 16 HRS or gt 32 HRS will impair the performance of the RNAscope Assay Dehydrate embed and cut the sample IMPORTANT Use fresh reagents 1 Wash sample with 1X PBS 2 Dehydrate sample using a standard ethanol series followed by xylene 3 Embed sample in paraffin using standard procedures Note Embedded samples may be stored at RT for years 4 Trim paraffin blocks as needed and cut embedded tissue into 5 1 ym sections using a microtome RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 11 5 Place paraffin ribbon in a 40 45 water and mount sections on SUPERFROST PLUS SLIDES Place tissue as shown for optimal staining Tissue section location 6 Air dry slides OVERNIGHT ot RT OPTIONAL STOPPING POINT Use sectioned tissue within 3 months Store sections with dessicants at RT RNAscope LS Assay for Leica Biosystems BOND RX System User
26. ol in the Name text box ACD Red in the Abbreviated name text box and ACD ISH Red Protocol in the Description text box RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 4 Select Bond Polymer Refine Red Detection under the Preferred detection system menu F BOND Protocol screen Hie don 2 New protocol properties ACD ISH Red Protocol Protocol type ACD Red ACD ISH Red Protocol RX1 Preferred detection system a Bond Polymer Refine Red Detection Step details ViewRNA Amp 1 v Incubetion time frin Wash Show wash stens v Double staining status 2 OETA Second cmm v 5 Highlight and select on each Reagent step to edit each step Set up the protocol steps highlighted rows according to the following table Step No Reagent Step Type Incubation Time Temperature 1 ACD Amp 1 Reagent 1 MIN 42 C 2 ACD Amp 1 Reagent 30 MIN 42 3 Bond Wash Solution Wash O MIN Ambient 4 Bond Wash Solution Wash O MIN Ambient 5 Bond Wash Solution Wash O MIN Ambient 6 Bond Wash Solution Wash 3 MIN Ambient 7 Bond Wash Solution Wash 3 MIN Ambient 8 Bond Wash Solution Wash O MIN Ambient 9 Bond Wash Solution Wash O MIN Ambient 10 Bond Wash Solution Wash O MIN Ambient 13 Bond Wash Solution Wash O MIN Ambient 14
27. omments Tissue type Dispense volume Testtissue NL tissue J 150 pL Postive tissue Staining mode Single D imn Process ou LE dapB 0 M Protocols Staining ACD ISH Red Protocol ls t 1 Bake and Dewax Y ACD 15min ER2 095 C v tsm ACD 15min Protease hl 2 H un v ACD 2 Hour Hybridization 8 Under the Preparation drop down menu select the protocol Bake and Dewax 9 Select Add slide for each target probe and the slides used for the run After adding all the slides to the study select Close to return to the Slide setup screen RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 29 30 D 10 Select Print labels to print barcodes that will be attached to the slides Run the RNAscope Assay Materials required Distilled Water Fume hood Xylene 100 EtOH Tissue Tek Staining Dish 4 Tissue Tek Clearing Agent Dish xylene resistant 2 Tissue Tek Vertical 24 Slide Rack EcoMount Cover Glass 24 mm x 50 mm Load the reagents 1 label each Leica BOND RX container with the corresponding reagent using water resistant marker 2 Use the barcode scanner to scan the barcode located on the front of each container including Leica Bond Refine Red and Hematoxylin kit A pop up menu will appear IMPORTANT NOT scan the barcode located on the top of each container 3 Choose the appropriate Reagent Name and enter the lo
28. on go to www acdbio com product_literature html At the website you can e Access telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support e Search for user documents MSDSs application notes citations training videos and other product support documents e Find out information about customer training events Contact information Advanced Cell Diagnostics Inc 3960 Point Eden Way Hayward CA 94545 Toll Free 1 877 576 3636 Direct 1 510 576 8800 Fax 1 510 576 8801 Information info acdbio com Orders orders acdbio com Support Email support acdbio com Limited product warranty Advanced Cell Diagnostics Inc and or its affiliate s warrant their products as set forth in the ACD General Terms and Conditions of Sale found on the ADC website at www acdbio com product_literature html f you have any questions please contact Advanced Cell Diagnostics at www acdbio com about contact RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 37 Headquarters 3960 Point Eden Way Hayward CA 94545 I Phone 1 510 576 8800 Toll Free 1 877 576 3636 AS For support email support acdbio com www acdbio com
29. probes in a 40 oven e Prepare two conical tubes of 30 ml of LS 1X Wash Buffer by adding 27 ml distilled water and 3 mL of LS Wash Buffer to each tube Mix well by inverting the tubes slowly at least five times Do not shake the tube Prepare the instrument Fill the large containers located in th reagents Dilute BOND Wash Solution 1 10 Note Insufficient bulk reagent volumes may lead to run failure bottom of the instrument with the Leica BOND RX bulk IMPORTANT Do not introduce bubbles into the solutions by shaking the containers To mix reagents gently invert the containers several times If bubbles are present leave the containers out at room temperature until the bubbles dissipate Use clean dry covertiles for every run Clean used covertiles with water bleach and ethanol Air dry before reuse Ensure waste bulk containers ar mptied before starting a run Discard waste according to all local state provincial and or national regulations RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red Register the reagents 1 Select the Reagent Setup icon at the top of the screen stat rim Lanni Lead ame Lanni Lama Lami Lamai rm Lami Lami Gani uasi Lama 2 Select Add to enter reagent information 3 Enter ACD Amp 1 in the Name text box 2 BOND RX
30. rvices guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cde gov biosafety e Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 2029cfr1910a_01 html e company s institution s Biosafety Program protocols for working with handling potentially infectious materials RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red 35 36 In the EU Additional information about biohazard guidelines is available at www ede gov Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety who cds csr lyo 2004 11 Information about the Registration Evaluation Authorisation and Restriction of Chemicals REACH can be found at eur lex europa eu LexUriServ LexUriServ do uri 0J L 2010 133 000 1 0043 EN PDF RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red Documentation and support Obtaining MSDSs Material Safety Data Sheets MSDSs are available at www acdbio com product_literature html For the MSDSs of chemicals not distributed by Advanced Cell Diagnostics contact the chemical manufacturer Obtaining support For the latest services and support informati
31. t 44 Bond Wash Solution Wash 1 MIN Ambient 45 Bond Wash Solution Wash 1 MIN Ambient 46 Bond Wash Solution Wash 1 MIN Ambient 49 Bond Wash Solution Wash O MIN Ambient 50 Bond Wash Solution Wash O MIN Ambient 5 Bond Wash Solution Wash O MIN Ambient 52 Bond Wash Solution Wash 1 MIN Ambient 53 Bond Wash Solution Wash 1 MIN Ambient 54 Bond Wash Solution Wash 1 MIN Ambient 55 Bond Wash Solution Wash 1 MIN Ambient 56 Bond Wash Solution Wash 1 MIN Ambient RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red Step No Reagent Step Type Incubation Time Temperature 59 Bond Wash Solution Wash O MIN Ambient 60 Bond Wash Solution Wash O MIN Ambient 61 Bond Wash Solution Wash O MIN Ambient 62 Bond Wash Solution Wash 1 MIN Ambient 63 Bond Wash Solution Wash 1 MIN Ambient 64 Bond Wash Solution Wash 1 MIN Ambient 65 Bond Wash Solution Wash 1 MIN Ambient 66 Bond Wash Solution Wash 1 MIN Ambient 71 De ionized Water Wash O MIN Ambient 72 De ionized Water Wash O MIN Ambient 73 De ionized Water Wash O MIN Ambient 74 De ionized Water Wash O MIN Ambient 75 De ionized Water Wash O MIN Ambient 76 De ionized Water Wash O MIN Ambient 77 Hematoyin Reagent 5MN Ambient 78 De ionized Water Wash O MIN Ambient 79 De ionized Water Wash O MIN Ambient 80 De ionized Water Wash O MIN Ambient 8
32. t number and expiration date in their respective text boxes Select OK 4 Load the containers onto the reagent tray and slide the tray into the Leica BOND RX module RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red AD 1 Once the barcodes have been ottached to the slides add the slides to the slide tray with the label sides facing up Note Each tray can accommodate only one study If a different protocol is used it must be placed in a separate tray Only three different parameters may be used for a complete run for a total of 30 slides Start the run 2 covertile on top of each slide The rectangularshaped neck of the covertile should fit into the groove of the slide tray 3 Place the tray in the Leica Bond and press the button to load tray onto the machine 4 Once the slides have been scanned select the triangular PLAY button on the screen located under the start tray to start the run Alternatively rightclick on scanned label images and select Delayed Start to start the run at a future time IMPORTANT Before leaving the instrument unattended ensure that the instrument is running successfully Note The following sections may also be performed using an automated coverslipper Complete the run 1 After the run is complete press the button on the instrument to unload the slides 2 Place the slides onto the Tissue Tek Slide Rack and move the rack into a stai
33. ystem User Manval Red User supplied materials IMPORTANT Do not substitute other materials for the SuperFrost Plus Slides listed in the following table Description Supplier Cat No SuperFrost Plus Slides required Fisher Scientific 12 550 15 100 ethanol EtOH American Master Tech Scientific MLS ALREAGAL Xylene Fisher Scientific MLS X3P 1GAL 10 neutral buffered formalin NBF MLS Paraffin wax MLS PBS MLS Microtome MLS Drying oven capable of holding temperature at 60 MLS 1 C optional Water bath or incubator capable of holding MLS temperature at 40 1 C EcoMount Biocare Medical EM897L Tissue Tek Vertical 24 Slide Rack American Master Tech Scientific MLS LWSRA24 Tissue Tek Staining Dish 4 required American Master Tech Scientific MLS LWT4457EA aa Agent Dish xylene resistant American Master Tech Scientific MLS LWT4456EA Cover Glass 24 x 50 mm Fisher Scientific MLS 12 545 F Distilled water MLS Fume hood MLS RNAscope LS Assay for Leica Biosystems BOND RX System User Manval Red Major Laboratory Supplier in North America For other regions please check Catalog Numbers with your local lab supplier 2 Chapter 2 Before You Begin Prior to running the RNAscope IS Assay on your samples for the first time we recommend that you Become familiar with Leica Biosystems BOND RX Research Advanced Stai
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