20-Step IHC-Paraffin Protocol Example
A Practical Step-by-Step Guide to IHC-Paraffin Staining
Immunohistochemistry on paraffin-embedded tissues (IHC-P) is a widely used technique for visualising protein expression while preserving tissue morphology.
This 20-step protocol outlines the key stages of a typical IHC-Paraffin workflow, including tissue preparation, antigen retrieval, blocking, antibody incubation, detection, counterstaining, and mounting.
Accompanied by practical tips and optimisation advice, this guide can help researchers develop robust staining protocols and generate reproducible, high-quality immunohistochemistry results.
- Mount and section samples
- Heat sections on the specimen slide to improve adherence
- Remove paraffin and rehydrate the tissue
- (Optional) Perform heat induced or protease induced epitope retrieval
- Wash
- Block endogenous peroxidases/phosphatases (for enzymatic labels) and biotin (for biotin/avidin detection systems)
-
Wash 1x in phosphate buffered saline (PBS)
Use Tris-buffered saline (TBS) instead of PBS for detecting phosphoproteins or when using alkaline phosphatase (AP) conjugated antibodies - Block non-specific binding sites
- Wash 3x in PBS or TBS
- Incubate with primary antibody
- Wash 3x in PBS or TBS
- (Optional) Incubate with secondary antibody
- Wash 3x in PBS or TBS
- (Optional) Incubate with amplification reagent
- Wash 3x in PBS or TBS
- Incubate with DAB or other substrate solution (for enzymatic labels only)
- Wash in ddH2O
- Counterstain
- Dehydrate tissue sections (for organic mounting media only)
- 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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