20-Step IHC-Paraffin Protocol Example
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- 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)
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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