Tips and Tricks for your Immunohistochemistry Experiments
Overview
The “IHC-paraffin tips and tricks guide” provides a general overview of how to perform immunohistochemistry-paraffin staining experiments and the different protocol steps involved.
The guide also includes background information on the technique and hands-on-advice on the optimization of individual protocol steps; special focus is placed on antigen retrieval, primary and secondary antibody selection, counterstains and mounting of coverslips.
This guide is helpful to IHC novices and more advanced researchers who are looking for tips and suggestions regarding the improvement of individual protocol steps.
Immunohistochemistry and Staining of FFPE Tissues 20-Step IHC-Paraffin Protocol Example References and Trademark Acknowledgements
Tips for Key Protocol Steps
- Step 1 - Mount and Section Samples
- Step 2 - Heat Sections
- Step 3 - Remove Paraffin and Rehydrate the Tissue
- Tips for Step 4 – Perform Antigen/Epitope Retrieval
- Step 5 - Wash 1x
- Tips for Step 6 – Block Endogenous Enzymes and Biotin
- Tips for Step 7 – Wash
- Tips for Step 8 – Block Non-specific Binding Sites
- Step 9 - Wash 3x
- Tips for Step 10 – Incubate with Primary Antibody
- Step 11 - Wash 3x
- Tips for (Optional) Steps 12 and 14– Incubate with Secondary Antibody and/or Amplification Reagent
- Tips for Step 13 – Wash
- Tips for (Optional) Steps 12 and 14 – Incubate with Secondary Antibody and/or Amplification Reagent
- Step 15 - Wash 3x
- Tips for Step 16 – Incubate with DAB or Other Substrates
- Step 17 - Wash in ddH2O
- Tips for Step 18 – Counterstain
- Step 19 - Dehydrate Tissue Sections
- Tips for Step 20 – Mount Coverslip
Related Resources
- IHC application page
- IHC protocols
- IHC-paraffin tested antibodies
- “Mastering IHC experiments webinar”
- Immunohistochemistry - Paraffin Tips & Tricks
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