Overview
The following protocols outline standard immunohistochemistry procedures used in the Microscopy Core Facility. Select a section below to view step-by-step instructions.
Immunohistochemistry Protocols
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ABC Immunohistochemistry Protocol (Paraffin Sections)
Day 1: Deparaffinization and Tissue Rehydration
- Melt paraffin sections in an oven at 65°C for 30 minutes. Allow slides to cool to room temperature.
- Prepare citrate antigen retrieval buffer and begin heating to 92–94°C.
- Rehydrate samples:
- Xylene: 2 × 5 minutes
- 100% ethanol: 2 × 2 minutes
- 95% ethanol: 2 × 2 minutes
- 75% ethanol: 1 × 2 minutes
- 50% ethanol: 1 × 2 minutes
- dH2O: 2 × 5 minutes
- PBS (pH 7.3): 1 × 5 minutes
Antigen Retrieval
- Incubate slides in citrate buffer for 40 minutes at 92–94°C
- Allow slides to cool to room temperature
- Wash slides in dH2O: 3 × 5 minutes
- Draw a hydrophobic barrier around tissue sections using a PAP pen
Immunostaining
- PBS rinse: 4 × 5 minutes
- 0.5% BSA + 0.4% Triton X-100 rinse: 1 × 10 minutes
- 1% H2O2 wash: 1 × 15 minutes
- Repeat PBS rinse: 4 × 5 minutes
- Repeat BSA/Triton rinse: 1 × 10 minutes
- Dry around tissue sections and place slides in humidity chamber
- Apply blocking buffer: incubate 1.5–2 hours at room temperature
- Replace with primary antibody solution and incubate overnight
Day 2: Secondary Antibody and Detection
- PBS rinse: 4 × 5 minutes
- BSA/Triton rinse: 1 × 10 minutes
- Apply biotinylated secondary antibody: incubate 2 hours
- Prepare ABC reagent (30 minutes prior; follow correct order)
- PBS rinse: 4 × 5 minutes
- BSA/Triton rinse: 1 × 10 minutes
- Apply ABC reagent: incubate 1–1.5 hours
Color Development
- Rinse with Tris buffer: 4 × 5 minutes
- Apply VIP development solution and monitor (2–20 minutes)
- Stop reaction with dH2O
- Rinse with dH2O: 2 × 2 minutes
Dehydration and Mounting
- Dehydrate through ethanol series to xylene
- Apply coverslip using Cytoseal
- Allow slides to air dry overnight
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ABC Immunohistochemistry Protocol (Frozen Sections)
Day 1: Immunostaining
- Draw barrier using PAP pen
- PBS rinse: 4 × 5 minutes
- BSA/Triton rinse: 1 × 10 minutes
- H2O2 wash: 1 × 15 minutes
- Repeat PBS rinse: 4 × 5 minutes
- Repeat BSA/Triton rinse: 1 × 10 minutes
- Apply blocking buffer: incubate 1.5–2 hours
- Apply primary antibody and incubate overnight
Day 2: Secondary Antibody and Detection
- PBS rinse: 4 × 5 minutes
- BSA/Triton rinse: 1 × 10 minutes
- Apply secondary antibody: incubate 2 hours
- Prepare ABC reagent (follow correct order)
- PBS rinse: 4 × 5 minutes
- BSA/Triton rinse: 1 × 10 minutes
- Apply ABC reagent: incubate 1–1.5 hours
Color Development and Mounting
- Rinse with Tris buffer: 4 × 5 minutes
- Apply VIP solution and monitor color development
- Stop reaction with dH2O
- Dehydrate through ethanol series
- Apply coverslip and allow to dry
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Fluorescence Immunohistochemistry Protocol (Mounted Slides)
Day 1
- Draw barrier using PAP pen
- PBS rinse: 4 × 5 minutes
- BSA/Triton rinse: 1 × 10 minutes
- Apply blocking buffer: incubate 2 hours
- Apply primary antibody and incubate overnight
Day 2
- PBS rinse: 4 × 5 minutes
- BSA/Triton rinse: 1 × 10 minutes
- Apply fluorescent secondary antibody: incubate 2 hours
- Use low-light conditions after applying fluorescent reagents
- Rinse with PBS: 4 × 5 minutes
- Apply mounting medium and coverslip
- Seal coverslip and allow to dry
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Biotin-Streptavidin Fluorescent Protocol (Mounted Slides)
Day 1
- Draw barrier using PAP pen
- PBS rinse: 4 × 5 minutes
- BSA/Triton rinse: 1 × 10 minutes
- Apply blocking buffer: incubate 2 hours
- Apply primary antibody and incubate overnight
Day 2
- PBS rinse: 4 × 5 minutes
- BSA/Triton rinse: 1 × 10 minutes
- Apply secondary antibody: incubate 2 hours
- Apply streptavidin antibody: incubate 2 hours
- Use low-light conditions
- Rinse with PBS
- Apply mounting medium and coverslip
- Seal coverslip and allow to dry