Immunohistochemistry Accessory Kit
Contents
1. Concentrated anti Rabbit IHC Antibody 1 drop Ready to Use Antibody Diluent 2 ml Total volume 2 ml Rinse dropper bottle with di H2O after each use DAB Substrate Using the dropper bottle provided prepare Working DAB Substrate DAB Solution A 1 drop DAB Solution B 1 drop DAB Solution C 1 drop Distilled Water 2 5 ml Total volume 2 5 ml Prepare just prior to use Rinse dropper bottle with di H2O after each use Deparaffination A B Treat slides with Xylene 3 changes for 5 minutes each Drain slides for 10 seconds between changes Treat slides with 100 Reagent Alcohol 3 changes for 5 minutes each Drain slides for 10 seconds between changes Ouench Endogenous Peroxidase A Place slides in peroxidase guenching solution not provided 15 30 minutes B Place slides in distilled water 2 changes for 2 minutes each Retrieve Epitopes A Preheat Epitope Retrieval Buffer Place 200 ml of Epitope Retrieval Buffer Working Solution into container cover and place into steamer e g Cuisinart CRC 800 Heat to 90 96 C B Place rack of slides into hot Working Epitope Retrieval Buffer for 20 minutes Cover C Carefully remove container with slides from steamer and cool on bench uncovered for 20 minutes D Slowly add distilled water to further cool for 5 minutes Rinse slides with distilled water 2 changes for 2 minutes each Immunostaining Procedure A B Remove each slide from rack and circle tissue2. Description Size Catalog No Concentrated Epitope Retrieval Buffer Reduced pH 100 ml IHC 101A Ready To Use IHC Blocking 60 ml Solution IHC 101B Ready To Use IHC Antibody 100 ml Diluent IHC 101C Concentrated anti Rabbit IHC 1 5 ml Antibody IHC 101D Concentrated IHC Wash Solution 50 ml IHC 101E Concentrated DAB Substrate 250 slides IHC 101F Ready To Use IHC Hematoxylin 60 ml IHC 101G Ready To Use IHC Bluing Solution 60 ml IHC 101H Concentrated Epitope Retrieval Buffer High pH 100 ml IHC 101J 10 Immunohistochemistry Accessory Kit Quick Reference Guide REAGENTS PROCEDURES Perform all steps at room temperature INCUBATIION Peroxidase Ouenching Solution Not provided Place slides in peroxidase guenching solution Incubate 15 30 min For paraffin embedded tissues Add 3 ml of 30 Hydrogen Peroxide Place slides in distilled water 2 changes for 2 minutes each to 200 ml of Methanol Mix well Epitope Retrieval Buffer Add 4 ml of Concentrated Epitope Preheat 200 ml of Epitope Retrieval Buffer to 90 96 C Place rack of 20 min Retrieval Buffer to 196 ml of distilled water pH should be 5 8 6 2 slides into hot Epitope Retrieval Buffer Cover Incubate Epitope Retrieval may not be reguired for your antibody Remove slides Cool uncovered for 20 min Add distilled water for 5 min Rinse with distilled water 2 changes for 2 minutes each Hydrophobic Barrier Pen Not provided Circle each
3. Peroxide solution Ready To Use IHC Hematoxylin 60 ml 2 8 C Ready To Use IHC Bluing Solution 60 ml 2 8 C Other Reagents Needed o Xylene Reagent Alcohol Hydrogen Peroxide Methanol IN HCl IN NaOH Hydrophobic marking pen Mounting media and coverslips 0000000 Protocol for Staining Formalin Fixed Paraffin Embedded Tissue Sections Buffer and Reagent Preparation Peroxidase Ouenching Solution To prepare 200 ml of Ouenching Solution 30 Hydrogen Peroxide 3 ml Methanol 200 ml Total volume 203 ml Use within 4 hours of preparation Epitope Retrieval Buffer Reduced pH To prepare 200 ml of Epitope Retrieval Buffer Working Solution Concentrated Epitope Retrieval Buffer 4 ml Reduced pH Distilled Water 196 ml Total volume 200 ml pH should be 5 8 6 2 Adjust pH if necessary with IN HCI or IN NaOH OR To prepare 1 liter of Epitope Retrieval Buffer Working Solution Concentrated Epitope Retrieval Buffer 20 ml Reduced pH Distilled Water 980 ml Total volume 1000 ml pH should be 5 8 6 2 Adjust pH if necessary with IN HCI or IN NaOH Store at to 2 8 C Discard after 3 months IHC Wash Solution To prepare 1 liter of Working IHC Wash Solution Concentrated IHC Wash Solution 5 ml Distilled Water 995 ml Total volume 1000 ml Store at 2 8 C Anti Rabbit IHC Antibody Using the dropper bottle provided prepare Working anti Rabbit IHC Antibody for 10 slides
4. section with a hydrophobic barrier pen e g Liquid Blocker Super Pap Pen Flood slide with Working IHC Wash Solution Do not allow tissue sections to dry for the rest of the procedure Drain wash solution and apply 4 drops of Ready To Use IHC Blocking Reagent to each slide and incubate for 15 minutes Drain Blocking Reagent do not wash off the Blocking Reagent apply 200 ul of Working Primary Antibody solution to each slide and incubate for 1 hour a To prepare Working Primary Antibody For Bethyl IHC antibodies add 2 10 ul of antibody to 1 ml of Ready To Use IHC Antibody Diluent mix Suggested starting dilutions are 1 100 1 500 Optimal working dilutions for other antibodies should be determined experimentally by the investigator T o Zz v o Wash slides with Working IHC Wash Solution 3 changes for 5 minutes each Drain wash solution apply 4 drops of Working anti Rabbit IHC Antibody to each slide and incubate for 1 hour Wash slides with Working IHC Wash Solution 3 changes for 5 minutes each Drain wash solution apply 4 drops of Working DAB Substrate to each slide and develop for 5 10 minutes Check development with microscope Wash slides with Working IHC Wash Solution 3 changes for 5 minutes each Drain wash solution apply 4 drops of Ready To Use IHC Hematoxylin to each slide and stain for 1 3 minutes Increase time if darker counterstaining is desired Wash slides with Working IHC Wash Solution 2 3 ch
5. Immunohistochemistry Accessory Kit For Use with Primary Antibodies made in Rabbit Cat No IHC 101 User Manual Qs BEDES BETHYL LABORATORIES INC The Polyclonal Antibody Specialists www bethyl com 1 800 338 9579 Table of Contents 1 fan 4078 HA Denter dE ne SODNA S ze nA en lie 3 LEO EE 4 Other Reagents Needed x sa ss R Eer 4 Protocol for Staining Formalin Fixed Paraffin Embedded Tissue Sections 5 Buffer and Reagent Dreparapon nee 5 Peroxidase Quenching Solution imtitenneeresereeeeeeeneeneeeneeenee 5 Epitope Retrieval Buffer Reduced pl 5 IHC Wash Solitons stas aja ne sleep aaa denen esses 6 Anti Rabbit THC Antibody nena 6 DAB re 6 IR en E le E H Quench Endogenous Peroxidase nena 7 Retrieve D Oe EE H Immunostaining Procedure eerreoeneeonnneoeneeonneeeeneeennneenaeennneeenae enne T ee 9 Ki LO 9 Replacement Reagent List 05555 sina da cisessereseaseepensaesdbessess vastpeastevveesoey bg t d 10 Immunohistochemistry Accessory Kit Quick Reference Guide 11 Introduction Bethyl Laboratories Immunohistochemistry Accessory Kit provides the reagents and method for sensitive reliable and economical immunohisto chemical staining This Kit is designed for immunostaining formalin fixed paraffin embedded tissue sections with primary antibodies made in rabbit The Kit contains all the reagents reguired for the imm
6. anges for 2 minutes each Drain wash solution and apply 4 drops of Ready To Use IHC Bluing Solution to each slide for 1 2 minutes Rinse slides in distilled water Soak slides in 70 reagent alcohol 3 minutes with intermittent agitation Soak slides in 95 reagent alcohol 2 changes for 3 minutes each with intermittent agitation Soak slides in 100 reagent alcohol 3 changes for 3 minutes each with intermittent agitation Drain slides for 10 seconds between each change Soak slides in Xylene 3 changes for 3 minutes each with intermittent agitation Drain slides for 10 seconds between each change Apply 2 3 drops of non agueous mounting media to each slide and mount coverslip Lay slides on a flat surface to dry prior to viewing under microscope Use treated slides e g HistoBond to assure adherence of FFPE sections to slide Prior to deparaffinization heat slides overnight in a 60 C oven All steps in which Xylene is used should be performed in a fume hood For Epitope Retrieval a microwave or pressure cooker may be substituted for the steamer method Adjust times as necessary depending on conditions For the initial IHC run with a new primary antibody test tissues with and without Epitope Retrieval In some instances Epitope Retrieval may not be necessary The Ready To Use IHC Antibody Diluent provided in this kit is recommended for Bethyl Laboratories primary and secondary antibodies 200 ul is the recommended maximum volu
7. me to apply to a slide for full coverage Using more than 200 ul may allow solutions to wick off the slide and create drying artifacts For small tissue sections less than 200 ul may be used 5 minutes of development with DAB Substrate should be sufficient Do not develop for more than 10 minutes If 5 minutes of development causes background staining further dilution of the primary antibody may be necessary IHC Hematoxylin was developed to produce a light nuclear counterstain so as not to obscure the DAB staining Counterstain for 1 1 minutes for nuclear antigens Counterstain for 2 3 minutes for cytoplasmic and membranous antigens If darker counterstaining is desired increase time up to 10 minutes Warranty Products are warranted by Bethyl Laboratories Inc to meet stated product specifications and to conform to label descriptions when used handled and stored according to instructions Unless otherwise stated this warranty is limited to 1 year from date of sale Bethyl Laboratories sole liability for the product is limited to replacement of the product or refund of the purchase price Bethyl Laboratories products are supplied for research applications They are not intended for medicinal diagnostic or therapeutic use The products may not be resold modified for resale or used to manufacture commercial products without prior written approval from Bethyl Laboratories Inc Replacement Reagent List
8. ready to use Apply 4 drops of Ready To Use IHC Bluing Solution to each slide 1 2 min Incubate Rinse with distilled water Dehydrate Clear and Mount Bethyl Laboratories Inc www bethyl com 800 338 9579 866 597 6105 11 Rev 150203 12
9. tissue section with the hydrophobic barrier pen Working Wash Solution Add 5 ml of concentrated Wash Solution to Flood each slide with Working Wash Solution Drain 5 min 995 ml of distilled water IHC Blocking Reagent ready to use Apply 4 drops of Ready To Use Blocking Reagent to each slide 15 mn Incubate Drain Do not wash Primary Antibody Not provided Suggested starting dilutions are Apply 200 ul of diluted Primary Antibody to each slide Incubate 1 100 1 500 for Bethyl IHC antibodies Optimal working dilutions for Wash with Working Wash Solution 3 changes for 5 minutes each other antibodies should be determined experimentally by the Drain investigator anti Rabbit IHC Antibody Add 1 drop of concentrated anti Rabbit Apply 4 drops of Working anti Rabbit IHC Antibody to each slide 1 hour IHC antibody to 2 ml of Ready To Use Antibody Diluent for every 10 Incubate Wash with Working Wash Solution 3 changes for 5 minutes slides being stained each Drain DAB Substrate To 2 5 ml of distilled water add 1 Apply 4 drops of Working DAB Substrate to each slide Incubate 5 10 min drop of DAB Solution A mix add 1 drop of DAB Wash with Wash Solution 3 changes for 5 minutes Drain Solution B mix add 1 drop of DAB Solution C mix IHC Hematoxylin ready to use Apply 4 drops of IHC Hematoxylin to each slide Incubate Wash 1 3 min with Working Wash Solution 2 3 changes for 2 minutes each Drain THC Bluing Solution
10. unostaining procedure and will stain 250 slides For convenience the Kit includes Ready To Use reagents in dropper bottles and additional dropper bottles for preparation of working solutions from concentrated reagents The anti Rabbit IHC Antibody improves immunohistochemical applications by reducing the processing time relative to other widely used detection systems The use of the anti Rabbit IHC Antibody results in sensitivity and specificity comparable to or better than the commonly used biotin streptavidin or biotin avidin detection systems The anti Rabbit IHC Antibody is designed to eliminate the streptavidin or avidin HRP step reguired when using biotinylated secondary antibodies Since there is no biotin in the system there is no need to block for endogenous biotin Storage Stability Store reagents at 2 8 C All reagents are stable for a minimum of one year from date of receipt Kit Components The Immunohistochemistry Accessory Kit components guantities and storage conditions are as follows Component Quantity Storage Concentrated Epitope Retrieval Buffer Reduced pH 100 ml 2 8 C Ready To Use IHC Blocking Solution 60 ml 2 8 C Ready To Use IHC Antibody Diluent 100 ml 2 8 C Concentrated anti Rabbit IHC Antibody 1 5 ml 2 8 C Concentrated IHC Wash Solution 50 ml 2 8 C Concentrated DAB Substrate DAB Solution A buffer 2 5 ml 2 8 C DAB Solution B DAB solution each DAB Solution C
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