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IHC Protocol

1. The document outlines the protocol for immunohistochemistry (IHC) staining of paraffin embedded tissue sections. 2. The protocol involves deparaffinization of the sections, antigen retrieval by heating the slides in sodium citrate buffer, blocking non-specific binding with serum, incubation with primary and secondary antibodies, development with DAB, counterstaining, dehydration and mounting. 3. Additional protocols are provided for immunofluorescence staining of paraffin and frozen tissue sections which involve similar steps of antigen retrieval, blocking, primary/secondary antibody incubation, counterstaining and mounting.

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Pradeep Patil
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41 views

IHC Protocol

1. The document outlines the protocol for immunohistochemistry (IHC) staining of paraffin embedded tissue sections. 2. The protocol involves deparaffinization of the sections, antigen retrieval by heating the slides in sodium citrate buffer, blocking non-specific binding with serum, incubation with primary and secondary antibodies, development with DAB, counterstaining, dehydration and mounting. 3. Additional protocols are provided for immunofluorescence staining of paraffin and frozen tissue sections which involve similar steps of antigen retrieval, blocking, primary/secondary antibody incubation, counterstaining and mounting.

Uploaded by

Pradeep Patil
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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IHC Protocol (For Paraffin Sections)

1. Deparafinization a) Keep the chamber in Xylene for 20 min (10 min in each chamber). b) Transfer the chamber to 99% Ethanol and keep for 5 min*2. c) Transfer the chamber to 95% Ethanol and keep for 5 min. d) Transfer the chamber to 70% Ethanol and keep for 5 min. e) Wash the slides in tap water for 10 min. 2. Antigen Retrieval Prep. Of 10mM Sodium Citrate Buffer (Antigen Retrieval Solution) Weigh 2.94g of Tris sodium citrate dehydrate and dissolve in 800ml Millipore water. Adjust the pH to 6.0 with 1N Hcl and add 0.5ml Tween 20. Make the volume finally to 1 litre. Cooking Set the pressure cooker at high temperature with 3/4th of water full and lid open. Fill the slide chamber with antigen retrieval solution. Just before the whistle starts set the temperature at 1000C and keep for 20min exactly. Cool for 20 minutes in cold water bath. 3. Transfer slides to DW --- 5 minutes 4. Put slides in egg white solution ---- 20 minutes 5. Wash slides in other coupling jar using warm water --- 10 minutes (5x 2).
6. Treat with milk solution for 20 minutes

7. Wash slides in other coupling jar using warm water --- 10 minutes (5x 2).
8. 9. 10. 11. Keep slide in PBS Circle with PAP Add blocking serum from secondary host --- 30 minutes (_____________________________) Remove serum and add diluted primary antibody (__________________) overnight 4 degree Or 37 degree for 1 -4 hours 12. Wash with PBS (5 x 3) 13. Block with H2O2 in methanol (1% of pure means 3% from 30% stock) ----- 15 minutes 14. Wash with PBS (5 x 3) 15. Add secondary antibody for 40 minutes (__________________________________) 16. Wash with PBS (5 x 3) 17. (Try to use fresh ) Add ABC reagent --- 30 60 minutes 18. Wash with PBS (5 x 3) 19. Incubate with DAB (45 seconds best more time leads false positive) 20. Wash with tap water ---- 5 minutes 21. Counterstain with hematoxylin --- 30 seconds to 1 minute 22. Wash with tap water ---- 5 minutes 23. 70% ethanol --- 2 minutes 24. 95% ethanol --- 2 minutes 25. 99% ethanol --- (2 x 2) 26. Xylene 20 minutes (10 x 2) 27. And mount DPX

IF protocol for paraffins slides 1. Paraffin slides (deparaffinize) 2. Antigen retrieval (20 minutes) 3. Run Tap Water (2 x 10 minutes) 4. Wash with PBS T20 (2 x 10minutes) 5. Circle with PAP pen 6. Block with 3% BSA or horse serum or serum of 2ndary antibody ( hour to 1 hour). 7. Primary antibody diluted in blocking solution or PBS (1-3 hours RT or overnight 4C). 8. Wash with PBS T20 (2 x 10minutes). 9. Add secondary antibody (40 minutes) 10. Wash with PBS T20 (4 x 5 minutes) 11. Counterstain with DAPI 12. Mount with aqueous mounting media. 13. Wait hour and paint with nail polish. 14. Wait for 2 hours or allow them to dry overnight and observe. IF protocol for cryoslides 1. Fix slides with acetone methanol (chilled solution) 2. Run Tap Water (2 x 10 minutes) 3. Wash with PBS T20 (2 x 10minutes) 4. Circle with PAP pen 5. Block with 3% BSA or horse serum or serum of 2ndary antibody ( hour to 1 hour). 6. Primary antibody diluted in blocking solution or PBS (1-3 hours RT or overnight 4C). 7. Wash with PBS T20 (2 x 10minutes). 8. Add secondary antibody (40 minutes) 9. Wash with PBS T20 (4 x 5 minutes) 10. Counterstain with DAPI 11. Mount with aqueous mounting media. 12. Wait hour and paint with nail polish. 13. Wait for 2 hours or allow them to dry overnight and observe.

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