RNAScope® Multiplex Reagent Kit For Tissues
Contents
1. RNAScope Multiplex Reagent Kit for Tissues 16 Purchaser Notification Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Fax 1 760 602 6500 Email techsupport dlifetech com European Headquarters nchinnan Business Park 3 Fountain Drive Paisley PA4 9RF UK Phone 44 141 814 6100 Toll Free Phone 0800 269 210 Toll Free Tech 0800 838 380 Fax 44 141 814 6260 Tech Fax 44 141 814 6117 Email euroinfo dinvitrogen com Email Tech eurotech invitrogen com Japanese Headquarters LOOP X Bldg 6F 3 9 15 Kaigan Minato ku Tokyo 108 0022 Japan Phone 81 3 5730 6509 Fax 81 3 5730 6519 Email jpinfo invitrogen com Additional international offices are listed at www lifetechnologies com 20 November 2013 These high quality reagents and materials must be used by or directly under the supervision of a technically qualified individual experienced in handling potentially hazardous chemicals Read the Safety Data Sheet provided for each product other regulatory considerations may apply Obtaining Support For the latest services and support information for all locations go to www lifetechnologies com At the website you can e Access worldwide 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 techsupportfalifetech com e Search for us2. Clearing Agent Dish in the fume hood 2 5 Incubate the slides in xylene for 5 minutes at room temperature Agitate the slides by occasionally lifting the slide rack up and down in the Clearing Agent Dish 2 6 Remove the slide rack from the first xylene containing dish and immediately place in the second xylene containing Clearing Agent Dish in the fume hood 2 7 Repeat Step 2 5 2 8 Remove the slide rack from the second xylene containing dish and immediately place in the Staining Dish containing 100 ethanol 2 9 Incubate the slides in 100 ethanol for 1 minute at room temperature with agitation 2 10 Repeat Step 2 9 with fresh 100 ethanol 2 11 Remove the slides from the rack and place on absorbent paper with the section face up Air dry for 5 minutes at room temperature Optional Stopping Point 3 Air dry overnight at room temperature must use within 24 hours or proceed directly to pretreatment page 9 RNAScope Multiplex Reagent Kit for Tissues 8 Step 3 Pretreat samples Pretreatment reagent selection Materials required Equilibrate the equipment The RNAScope Multiplex Fluorescent Assay is compatible with fresh frozen FF tissue fixed frozen tissue formalin fixed paraffin embedded FFPE tissue cultured adherent cells on chamber slides and Peripheral Blood Mononuclear Cells PBMC IMPORTANT Different tissue and cell types have different pretreatment requirements Refer to Table 4 below to determine the
3. any of your samples to optimize the protocol Materials required Materials provided by the RNAScope Fluorescent Multiplex Kit e 50x Wash Buffer e Amp 1 FL e Amp 2 FL e Amp 3 FL e Amp 4 Alt A FL Amp 4 Alt B FL or Amp 4 Alt C FL e DAPI Materials provided by RNAScope Probes e C1 Target Probe e 50x C2 Target Probe e 50x C3 Target Probe e 3 Plex Positive Control Probe e Negative Control Probe Other materials and equipment e Prepared sections e Distilled water e Carboy gt 3L e Tissue Tek Staining Dish e Tissue Tek Clearing Agent Dish xylene resistant e Hybridization oven e Water bath or incubator e Tissue Tek Vertical 24 Slide Rack e Tubes various sizes e Paper towel or absorbent paper e ProLong Gold Antifade Reagent 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 the appropriate sample preparation and pretreatment sections of this user guide Some of the materials may be prepared in advance and stored at room temperature RNAScope Multiplex Reagent Kit for Tissues 11 Prepare 1x Wash Buffer 4 1 Prepare 3 L of 1x Wash Buffer by adding 2 94 L distilled water and 1 bottle 60 mL of 50x Wash Buffer to a large carboy Mix well Note Warm 50x Wash Buffer up to 40 C for 10 20 minutes before diluting it to prepare 1x Wash Buffer 1x Wash Buffer may be prepared ahead of time and stored at room tempe
4. chamber slides or Peripheral Blood Mononuclear Cells PBMC see Table 4 page 9 If you are using other sample types skip Steps 3 13 3 16 3 13 Place the dried slides on the hybridization slide rack and add 5 drops of Pretreat 2 reagent to entirely cover each section 3 14 Incubate for 30 minutes at room temperature 3 15 Take each slide one at a time from the hybridization slide rack and tap and or flick to remove the excess liquid Immediately place each slide in a Tissue Tek Slide Rack submerged in a Tissue Tek Staining Dish filled with 1x PBS 3 16 Wash slides with 1x PBS by moving the rack up and down 3 5 times and repeat with 1x PBS Apply Pretreat 4 reagent Application of Pretreat 4 reagent is required fixed frozen and FFPE tissues already pretreated with 1x Pretreat 2 reagent or for untreated fresh frozen FF tissues see Table 4 page 9 3 17 Follow the same procedure as the application of Pretreat 3 reagent Steps 3 13 3 16 but use Pretreat 4 reagent instead RNAScope Multiplex Reagent Kit for Tissues 10 Step 4 Run the RNAScope Fluorescent Multiplex Assay This procedure flows directly from sample preparation and pretreatment Refer to the appropriate sample preparation and pretreatment sections of this user guide for your specific sample type IMPORTANT Do not let sections dry out between incubation steps Work quickly and fill barrier with solutions Note We recommend running controls before running
5. housekeeping genes 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 six months from the shipment date when stored at 2 8 C The RNAScope Probes consist of user specified Target Probes and Positive and Negative Control Probes Each Target Probe contains a mixture of short oligonucleotides designed to bind to a specific target RNA and is detectable in one of three color channels C1 C2 and C3 using the Amp 4 amplification step Channel C1 target probes are Ready To Use RTU while channel C2 and C3 probes are shipped as a 50x concentrated stock To independently detect different target RNAs in a multiplex assay each target probe must be in a different color channel and there must be a C1 probe in the mixture If no specific C1 probe is used then a Blank Probe C1 can be used in place of a specific target probe Different colors are assigned to the C1 and C2 color channels depending on the particular RNAScope Assay You can select any combination based on your imaging instrument configuration For example a higher expression gene on a lower wavelength i e green channel The color channels for the RNAScope Multiplex Fluorescent Assay are shown in Table 3 below There are 3 options for alternate fluorescent color modules Any fluorescent label combination Amplificatio
6. 9 prepare 700 mL of fresh 1x Pretreat 2 reagent by adding 630 mL of distilled water to 1 bottle of 10x Pretreat 2 solution 70 mL ina 1 L beaker Mix well 3 6 Place the beaker containing 1x Pretreat 2 reagent on the hot plate Cover the beaker with foil and turn the hot plate on high for 10 15 minutes 3 7 Once 1x Pretreat 2 reagent reaches boiling turn the hot plate knob to 100 104 C to maintain uniform boiling IMPORTANT Do not boil the 1x Pretreat 2 reagent for more than 30 minutes before use Apply Pretreat 2 reagent Application of Pretreat 2 reagent is required only for fixed frozen and FFPE tissues see Table 4 page 9 3 8 Ensure that 1x Pretreat 2 solution is at mild boiling 3 9 With a pair of forceps very slowly submerge the slide rack containing the slides into the boiling 1x Pretreat 2 solution Cover the beaker with foil and boil the slides for 10 30 minutes 3 10 After pretreatment time is over use the forceps to immediately transfer the hot slide rack from the 1x Pretreat 2 solution to the Staining Dish containing distilled water Do not let the slides cool in Pretreat 2 3 11 Wash slides in the distilled water by moving the rack up and down 3 5 times and repeat with fresh distilled water 3 12 Wash slides in a dish filled with fresh 100 ethanol by moving the rack up and down 3 5 times Air dry the slides Apply Pretreat 3 reagent Application of Pretreat 3 reagent is required only for cultured adherent cells on
7. OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF Important Licensing Information These products may be covered by one or more Limited Use Label Licenses By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses The trademarks mentioned herein are the property of Life Technologies Corporation and or its affiliate s or their respective owners RNAScope is a registered trademark of Advanced Cell Diagnostics Inc ImmEdge is a trademark of Vector Laboratories Ltd SuperFrost and SuperFrost Plus are registered trademarks of Liebherr International AG Tissue Tek is a registered trademark of Sakura Finetek Kabushiki Kaisha Parafilm is a registered trademark of Bemis Company Inc Kimwipes is a registered trademark of Kimberly Clark Corporation 2013 Life Technologies Corporation All rights reserved technologies
8. PreAMP AMP Label probes gt Zz z z E z CE ZZ zZz ZZ 1 Tissue section 2 Hybridize to target RNA 3 Amplify signal 4 Image Start with properly prepared Hybridize multiple sets of Use up to 4 signal amplification systems Visualize target RNA tissue sections and prereat to gene specific probe pairs to detect multiple target RNAs Probes are using standard allow access to target RNA to target mRNAs hybridized to a cascade of signal amplification fluorescent microscope molecules culminating in binding of dye labeled probes visible in different fluorescent channels RNAScope Multiplex Reagent Kit RNAScope Multiplex Reagent Kits contain three modules a Pretreatment Module a Detection Module and a Wash Buffer Module see Table 1 page 1 When stored as directed the reagents are stable at least until the expiration date printed on the product label e The Pretreatment Module consists of Pretreat 2 Pretreat 3 and Pretreat 4 reagents which are used in different combinations to pretreat various tissue types see Pretreatment reagent selection page 9 to prepare them for the RNAScope in situ hybridization protocol e The Detection FL Module contains the propriatery signal amplification and detection reagents that hybridize to the gene specific RNA probes i e Target Probes available separately to detect multiple target RNAs e The Wash Buffer Module contains the 50x RNAScope Wash Buffer th
9. appropriate pretreatment reagent Table 4 Pretreatment selection guide for fluorescence detection Tissue type Fresh frozen tissue Pretreatment reagent Pretreatment 4 only Fixed frozen tissue Pretreatment 2 and Pretreatment 4 FFPE tissue Pretreatment 2 and Pretreatment 4 Cultured adherent cells Pretreatment 3 only Peripheral Blood Mononuclear Cells PBMC Pretreatment 3 only Note Pretreatment with Pretreat 2 reagent reagent is required only for fixed frozen and FFPE tissues If you are using fresh frozen FF tissue cultured adherent cells on chamber slides or Peripheral Blood Mononuclear Cells PBMC skip Steps 3 5 3 12 page 10 e Pretreat 2 3 and 4 reagents from the Pretreatment Module of the RNAScope Multiplex Reagent Kit e Prepared slides e Distilled water e Paper towel or absorbent paper e Hybridization oven e Tissue Tek Slide Rack e Tissue Tek Staining Dish e Distilled water e Glass beaker 1 or 2 L e Hot plate e Aluminum foil e Thermometer e Forceps large 3 1 Turn on the hybridization oven and set the temperature to 40 C 3 2 Place a humidifying paper in the humidity control tray and wet completely with distilled water 3 3 Insert covered tray into oven and close the oven door Warm the tray for 30 minutes at 40 C before use RNAScope Multiplex Reagent Kit for Tissues 9 Prepare 1x Pretreat 2 reagent 3 5 If required see Table 4 page
10. at supports the RNAScope assay workflow Each RNAScope Multiplex Reagent Kit provides enough reagents to stain 20 tissue sections of approximately 20 mm x 20 mm in size Larger tissue sections will result in fewer tests Note that in addition to the RNAScope Multiplex Reagent Kit RNAScope Assays require gene specific RNA probes RNAScope Probes page 3 which are available separately from Life Technologies IMPORTANT RNAScope Reagent Kits share the same Pretreatment Module and Wash Buffer but have unique Detection Modules Do not interchange the reagent components of the Detection Modules even those having the same name RNAScope Multiplex Reagent Kit for Tissues 2 Sample types RNAScope Probes To perform the RNAScope Assay you must start with properly prepared and pretreated samples Multiple sample types are compatible with RNAScope Assays and include formalin fixed paraffin embedded FFPE tissues fresh frozen tissues fixed frozen tissues tissue microarray TMA and cell samples RNAScope Probes are gene specific RNA probes that are required for RNAScope Assay but are not included in the RNAScope Reagent Kit Based on the gene of interest these probes can be ordered separately from Life Technologies Contact us to find a gene specific probe from a searchable catalog of gt 27 000 pre designed Target Probes or order a custom probe Note that pre designed control probes are also available for
11. des IMPORTANT Place sections in the center of the slide 1 8 Air dry the slides overnight at room temperature Do not bake slides unless they will be used within 1 week Optional Stopping Point 1 This is an optional stopping point in the procedure Use the sectioned tissue within 3 months Store sections with dessicants at room temperature RNAScope Multiplex Reagent Kit for Tissues 7 Step 2 Prepare FFPE slides for the RNAScope Assay Materials required e Drying oven e Prepared FFPE slides e Tissue Tek Vertical 24 Slide Rack e Distilled water e Fume hood e Xylene e 100 ethanol e Tissue Tek Clearing Agent Dish 2 e Tissue Tek Staining Dish 2 e ImmEdge Hydrophobic Barrier Pen Bake slides 2 1 Bake slides in a dry oven for 1 HOUR at 60 C Optional Stopping Point 2 Use sectioned tissue within 1 week Store sections with dessicants at room temperature 2 2 If you wish you continue prepare the materials for deparaffinizing the FFPE sections see below and pretreating the samples see page 9 while the slides are baking Deparaffinize FFPE sections 2 3 In a fume hood fill two dishes designated as Clearing Agent Dishes with 200 mL of fresh xylene and two dishes designated as Staining Reagent Dishes with 200 mL of fresh 100 ethanol Note Reagents may be prepared ahead of time Ensure all containers remain covered 2 4 Place slides in a Tissue Tek Slide Rack and submerge in the first xylene containing
12. e slide at a time quickly remove excess liquid by decanting and place slide in a Tissue Tek Slide Rack submerged in the Tissue Tek Staining Dish filled with 1x Wash Buffer 4 20 Wash slides in 1x Wash Buffer for 2 minutes at room temperature with occassional agitation 4 21 Repeat Step 4 20 with fresh 1x Wash Buffer Hybridize Amplification Reagent 3 FL 4 22 Take each slide one at a time from the Tissue Tek Slide Rack and tap and or flick to remove the excess liquid Add 4 drops of Amplification Reagent 3 FL to entirely cover each section 4 23 Incubate the slides for 30 minutes at 40 C in the hybridization oven Make sure that the humidity is controled in the chamber and do not let the slides dry out 4 24 Remove one slide at a time quickly remove excess liquid by decanting and place slide in a Tissue Tek Slide Rack submerged in the Tissue Tek Staining Dish filled with 1x Wash Buffer 4 25 Wash slides in 1x Wash Buffer for 2 minutes at room temperature with occassional agitation 4 26 Repeat Step 4 25 with fresh 1x Wash Buffer RNAScope Multiplex Reagent Kit for Tissues 13 Hybridize Amplification Reagent 4 FL 4 27 Take each slide one at a time from the Tissue Tek Slide Rack and tap and or flick to remove the excess liquid Add 4 drops of Amplification Reagent 4 FL to entirely cover each section Note There are 3 options for alternate fluorescent color modules Any fluorescent label combination Amplifica
13. er documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents e Obtain information about customer training e Download software updates and patches SDS Safety Data Sheets SDSs are available at www lifetechnologies com sds Certificate of Analysis The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the product packaging tube pouch or box Limited Product Warranty Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support Disclaimer LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE
14. erged in the Tissue Tek Staining Dish filled with 1x Wash Buffer 4 10 Wash slides in 1x Wash Buffer for 2 minutes at room temperature Agitate slides by moving the Slide Rack up and down in the dish 4 11 Repeat Step 4 10 with fresh 1x Wash Buffer RNAScope Multiplex Reagent Kit for Tissues 12 Hybridize Amplification Reagent 1 FL 4 12 Take each slide one at a time from the Tissue Tek Slide Rack and tap and or flick to remove the excess liquid Add 4 drops of Amplification Reagent 1 FL to entirely cover each section 4 13 Incubate the slides for 30 minutes at 40 C in the hybridization oven Make sure that the humidity is controled in the chamber and do not let the slides dry out 4 14 Remove one slide at a time quickly remove excess liquid by decanting and place slide in a Tissue Tek Slide Rack submerged in the Tissue Tek Staining Dish filled with 1x Wash Buffer 4 15 Wash slides in 1x Wash Buffer for 2 minutes at room temperature with occassional agitation 4 16 Repeat Step 4 15 with fresh 1x Wash Buffer Hybridize Amplification Reagent 2 FL 4 17 Take each slide one at a time from the Tissue Tek Slide Rack and tap and or flick to remove the excess liquid Add 4 drops of Amplification Reagent 2 FL to entirely cover each section 4 18 Incubate the slides for 15 minutes at 40 C in the hybridization oven Make sure that the humidity is controled in the chamber and do not let the slides dry out 4 19 Remove on
15. ess tissue and cell morphology e Assess positive control signal strength Positive control signal should be visible as punctuate dots within cell e Assess negative control background One dot in every 10 cells displaying back ground staining per microscope field is acceptable e Evaluate target probe signal using the scoring guidelines in the next section Figure 2 below is an example showing expression in the cerebral cortex of normal mouse brain Figure 2 Npy red and Fezf2 green expression in the cerebral cortex of normal mouse brain stained using the RNAScope Fluorescent Multiplex Kit 63x oil lens confocal image RNAScope Multiplex Reagent Kit for Tissues 15 Appendix A Reagent Volume Guidelines Table 5 Reagent volume guidelines Before starting your experiment measure the inner edge of the hydrophobic barrier to determine the recommended number of drops needed per slide see Table 5 below Size of hyrophobic barrier Recommended number of Recommended volume per 4 in drops per slide slide uL ee 0 75 x 0 75 f 4 120 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
16. etreatment see page 7 For the latest protocols and guidelines using other sample types and preparation methods contact Technical Support IMPORTANT We highly recommend following these guidelines We cannot guarantee assay results with other preparation methods Scalpel Forceps Cryo embedding medium OCT Dry ice liquid nitrogen or isopentane Cryostat Slide boxes SuperFrost Plus slides Aluminum foil or zip lock bags 1 1 Remove tissue and cut to fit into cryomolds CAUTION Handle biological specimens appropriately 1 2 Freeze the specimen on dry ice or in liquid nitrogen within 5 minutes of tissue harvest RNAScope Multiplex Reagent Kit for Tissues 4 1 3 Embed frozen tissue in cryo embedding medium OCT a Add two drops of OCT into a cryomold b Place the frozen tissue on the OCT in the correct orientation for cutting c Add more OCT to fill the cryomold Do not allow any air bubbles to form d Hold the block with forceps on the surface of liquid nitrogen or isopentane cooled by dry ice or liquid nitrogen or place the cryomold on dry ice 1 4 Store the frozen block in an air tight container at 80 C prior to sectioning Embedded tissue may be stored for at least 3 months Section the block 1 5 Equilibrate block to 20 C in a cryostat for 1 hour 1 6 Cut 10 20 pm sections and mount onto SuperFrost Plus slides 1 7 Keep the sections at 20 C to dry 1 8 Store the sections in slide boxes wrapped air t
17. f brought to you by e A D lik technologies RNAScope Multiplex Reagent Kit For Tissues Catalog no R26963 Table 1 Contents and storage Component Amount Storage Pretreatment Module Pretreat 2 reagent 10x 4x70 mL Room temperature Pretreat 3 reagent 1x 4 5 mL 2 8 C Pretreat 4 reagent 2x 4 5 mL Wash Buffer Module Washing Buffer 50x 4x 60 mL Room temperature Detection FL Module Amplification Reagent 1 FL 1x 3mL Amplification Reagent 2 FL 1x 4 5 mL Amplification Reagent 3 FL 1x 3mL Amplification Reagent 4 FL Alt A Display module 1x 4 5 mL 2 8 C Amplification Reagent 4 FL Alt B Display module 1x 4 5 mL Amplification Reagent 4 FL Alt C Display module 1x 4 5 mL DAPI 1x3mL Each RNAScope Reagent Kit provides enough reagents to stain 20 tissue sections of approximately 20 mm x 20 mm in size Larger tissue sections will result in fewer tests When stored as directed the reagents are stable at least until the expiration date printed on the product label Table 2 Gene specific RNAScope Probes not included Component Amount Storage Ready To Use RTU probe for color channel 1 1x3mL 50x probe for color channel 2 1x 60 uL 2 8 C 50x probe for color channel 3 1x 60 uL Gene specific custom RNAScope Probes are required for the RNAScope Assay but are not included in the RNAScope Multiplex Reagent Kit Based on the gene of i
18. ight with aluminum foil or zip lock bags at 80 C until use Sections may be stored for at least 3 months IMPORTANT Do not fix the slides prior to this step Optional Stopping Point 1 This is an optional stopping point in the procedure Use the sectioned tissue within 3 months Step 2 Prepare FF slides for the RNAScope Assay Materials required e 1x PBS e 10 neutral buffered formalin NBF e 100 ethanol e Tissue Tek Vertical 24 Slide Rack e Tissue Tek Staining Dishes 5 required e ImmEdge Hydrophobic Barrier Pen Fix the sections 2 1 Chill 200 mL 10 NBF fresh 10 NBF or 4 paraformaldehyde in 1X PBS to 4 C ina Tissue Tek Staining Dish 2 2 Remove slides from 80 C and place in a Tissue Tek Slide Rack 2 3 Immediately immerse slides in the pre chilled fixative Fix for 15 minutes at 4 C Note Formalin that has been stored for more than 6 months exposed to air for more than a week or used repeatedly may result in suboptimal tissue fixation RNAScope Multiplex Reagent Kit for Tissues 5 Dehydrate the sections Reagents may be prepared ahead of time Ensure all containers remain covered 2 4 Prepare 200 mL 50 ethanol 200 mL 70 ethanol and 600 mL 100 ethanol in Tissue Tek Staining Dishes 2 5 Place the slides in 50 ethanol for 5 minutes at room temperature 2 6 Place the slides in 70 ethanol for 5 minutes at room temperature 2 7 Place the slides in 100 ethanol for 5 minutes at room temperatu
19. lin fixed paraffin embedded FFPE sections Prepare formalin fixed paraffin embedded FFPE sections as described below For fresh frozen FF sample preparation and pretreatment see page 4 For the latest protocols and guidelines using other sample types and preparation methods contact Technical Support IMPORTANT We highly recommend following these guidelines We cannot guarantee assay results with other preparation methods Materials required e 10 neutral buffered formalin NBF e 1x PBS e Paraffin wax e 100 ethanol ACS grade or equivalent e Xylene e Microtome e Water bath e SuperFrost Plus slides Fix the sample 1 1 Remove sample and cut 3 4 mm pieces prior to fixing CAUTION Handle biological specimens appropriately 1 2 Fix sample in fresh 10 NBF for 16 32 hours at room temperature Fixation time will vary depending on tissue type IMPORTANT Under fixation will result in significant signal loss when performing the RNAScope Assay Dehydrate embed and cut the sample IMPORTANT Use fresh reagents 1 3 Wash sample with 1x PBS 1 4 Dehydrate the sample using a standard ethanol series followed by xylene 1 5 Embed the sample in paraffin Note Embedded samples may be stored at room temperature for years 1 6 Trim paraffin blocks as needed and cut embedded tissue into 5 pm 1 um sections using a microtome 1 7 Place paraffin ribbon in water bath at room temperature and mount sections on SuperFrost Plus sli
20. n Reagent 4 Alt A B or C can be selected based on your experiment design Table 3 Color module options for the RNAScope Multiplex Fluorescent Assay Fluorescent label C1 Color Ex Em C2 Color Ex Em C3 Color Ex Em Amplification Reagent 4 Alt A FL Green 488 540 nm Orange 550 580 nm Far red 647 690 nm Amplification Reagent 4 Alt B FL Orange 550 580 nm Green 488 540 nm Far red 647 690 nm Amplification Reagent 4 Alt C FL Orange 550 580 nm Far red 647 690 nm Green 488 540 nm RNAScope Multiplex Reagent Kit for Tissues 3 Before starting Important guidelines Start with properly prepared sections Refer to the sample preparation and pretreatment sections of this user guide Use only samples mounted on SuperFrost Plus Slides Fisher Scientific Cat no 12 550 15 Always run positive and negative control probes on your sample to assess sample RNA quality and optimal permeabilization Do not substitute required materials RNAScope Assays have 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 Step 1 Prepare fresh frozen FF sections Materials required Prepare the block Prepare fresh frozen FF sections as described below For formalin fixed paraffin embedded FFPE sample preparation and pr
21. nterest they can be ordered separately from Life Technologies which offers over 27 000 pre designed probes If the required probe for the RNA of interest can not be found among the pre designed probes then a design fee will be charged Positive and negative probes for common housekeeping are also available for ordering For Research Use Only Not for use in diagnostic procedures MANO0009813 MP26963 Revision A 0 Introduction The RNAScope Assays use a novel and proprietary method of in situ hybridization ISH to visualize single RNA molecules per cell in samples mounted on slides The assays are based on the patented signal amplification and background suppression technology Proprietary RNA specific probes RNAScope Probes available separately are hybridized to target RNA and are then bound to a cascade of signal amplification molecules culminating in signal detection Figure 1 below Single plex 2 plex multiplex and automated assays are all available The RNAScope Assay procedure is illustrated in Figure 1 below and can be completed in 6 10 hours depending on the assay or conveniently divided over two days Most RNAScope Assay reagents are available in convenient Ready To Use RTU dropper bottles and provide a simple nearly pipette free workflow Results are observable using standard bright field or fluorescent microscopy Figure 1 Overview of the RNAScope Assay procedure Z Z Target RNA specific oligo probes
22. rature for up to one month Prepare RNAScope Probes 4 2 Warm the RNAScope Probes for 10 minutes at 40 C in a water bath or incubator then cool to room temperature 4 3 Briefly spin the C2 and C3 probes to collect the liquid at the bottom of the tubes 4 4 Mix the C2 C3 and C1 probes at a ratio of 1 1 50 by pipetting 1 volume of C2 and 1 volume of C3 probes into 50 volumes of C1 probe in a sterile tube Invert the tube several times Note Do not mix probes of the same channel Mixed Target Probes can be stored at 4 C for up to 6 months Equilibrate reagents 4 5 Place AMP 1 4 FL reagents at RT 4 6 Make sure that the hybridization oven and the prepared humidity control tray are at 40 C Hybridize probe IMPORTANT Make sure that RNAScope Probes are prewarmed and cooled to room temperature prior to use 4 7 Tap and or flick to remove excess liquid from slides and add 4 drops of the appropriate probe to entirely cover each section Note Refer to Appendix A Reagent Volume Guidelines on page 16 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 4 8 Place slides in the hybridization oven and incubate for 2 hours at 40 C Make sure that the humidity is controled in the chamber and do not let the slides dry out 4 9 Remove one slide at a time quickly remove excess liquid by decanting and place slide in a Tissue Tek Slide Rack subm
23. re 2 8 Repeat step 2 7 with fresh 100 ethanol Optional Stopping Point 2 This is an optional stopping point in the procedure Slides may be stored in 100 ethanol at 20 C for up to 1 week Prolonged storage may degrade sample RNA Create a hydrophobic barrier 2 9 Take slides out of 100 ethanol and place on absorbent paper with the section face up Air dry for 5 minutes at room temperature 2 10 Using the following example draw a barrier 2 4 times around each section with the ImmEdge hydrophobic barrier pen See example below size of this hydrophobic barrier is 0 75 x 0 75 Tissue Slide Label Note Refer to Appendix A Reagent Volume Guidelines on page 16 to determine the recommended number of drops needed per slide IMPORTANT Do not let the barrier touch the section InmEdge hydrophobic barrier pen is highly recommended Alternative type of pen may result in suboptimal results Note We do not recommend drawing a smaller barrier and using less than the recommended volume amounts even for smaller sections Larger barriers will result in fewer tests per kit 2 11 Let the barrier dry completely for 1 minute Note If you need to reapply the hydrophobic barrier during pretreatment dry the appropriate area of the slide with a Kimwipes laboratory tissues Do not touch the tissue section 2 12 Proceed to pretreatment page 9 RNAScope Multiplex Reagent Kit for Tissues 6 Step 1 Prepare forma
24. tion Reagent 4 FL Alt A B or C can be selected For fresh frozen sections module Amp 4 Alt B is recommended for all other sample types Amp 4 Alt A is recommended 4 28 Incubate the slides for 30 minutes at 40 C in the hybridization oven Make sure that the humidity is controled in the chamber and do not let the slides dry out 4 29 Remove one slide at a time quickly remove excess liquid by decanting and place slide in a Tissue Tek Slide Rack submerged in the Tissue Tek Staining Dish filled with 1x Wash Buffer 4 30 Wash slides in 1x Wash Buffer for 2 minutes at room temperature with occassional agitation 4 31 Repeat Step 4 30 with fresh 1x Wash Buffer Counterstain and mount the slides IMPORTANT Do not perform this procedure with more than 5 slides at a time 4 32 Remove excess liquid from the slides and add 4 drops of DAPI to each section 4 33 Incubate for 30 seconds at room temperature 4 34 Remove DAPI from slides and immediately place 1 2 drops of ProLong Gold Antifade Reagent onto each section 4 35 Carefully place a 24 mm x 50 mm coverslip over the tissue section Avoid trapping air bubbles Store slides in the dark at 4 C RNAScope Multiplex Reagent Kit for Tissues 14 Step 5 Evaluate the samples Control examples For an example of successful staining see Figure 2 Examine tissue sections under a standard fluorescent microscope at 20 40x magnification A confocal microscope may also be used e Ass
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