Histology Immunohistochemistry

Antigen retrieval

• 0.1 M citric acid (21 g in 1L)
• 0.1 M sodium citrate (29.4 g in 1L)
• Prepare 9 mL of citric acid in 41 mL sodium citrate and make up to 500 mL with water.
• Bring to 90 degrees C by heating in a microwave
• Mircowave sections for 10 minutes; cool at room temperature for 20 minutes.

Immunohistochemistry (Avidin-Biotin complex method)

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• Dewax slides in histoclear for 5 minutes, then dehydrate in graded ethanol solutions (2x100% and 1X70% ethanol) for 1 min. in each solution.
• Wash in running tap water for 1 min.
• Perform antigen retrieval if necessary.
• Outline with Dako pen; keep moist with PBS.
• Block endogenous peroxidase by incubating with 6% H2O2 for 10 min. at room temp.
• Wash with PBS 3 x 5 min.
• Block endogenous Avidin and Biotin (15 min. each with a PBS rinse in between steps).
• Block non-specific labeling by incubating with 10% normal goat serum (in PBS) for 20 min. at room temp. Note: if the primary antibody is raised in goat, use 0.1% albumin as a blocking step.
• Tip off serum (except negative control) and incubate with primary antibody O/N at 4 degrees C or 2 hours at room temperature (dilution of Ab varies).
• Wash 3x 5 min. in PBS.
• Incubate with secondary antibody (1:100 dilution) for 1 hour at room temp.
• Wash with PBS 3 x 5 min.
• Apply ABC 1:100 for 1 hour at room temp. (Note: prepare at least 30 minutes before use)
• Wash with PBS 3 x 5 min.
• Apply DAB until color develops. (Always wear gloves for this, it's potentially carcinogenic.)
• Wash with water (then neutralize water with bleach before tipping down the drain).
• Counterstain with hematoxylin for 30 secs.
• Wash with running tap water.
• 2 dips in 70% ethanol.
• 4 dips in 100% ethanol.
• 1 minute in 100% ethanol.
• 1 minutes in histoclear.
• 5 minutes in histoclear.
• Mount in DePeX.

Immunofluorescence

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• Dewax slides in histoclear for 5 minutes, then dehydrate in graded ethanol solutions (2x100% and 1X70% ethanol) for 1 minute in each solution.
• Wash in running tap water for 1 minute.
• Perform antigen retrieval if necessary.
• Outline with Dako pen; keep moist with PBS.
• Block non-specific labeling by incubating with 10% normal goat serum (in PBS) for 20 min. at room temp. Note: if the primary antibody is raised in goat, use 0.1% albumin as a blocking step.
• Tip off serum (except negative control) and incubate with primary antibody O/N at 4 degrees C or 2 hours at room temp. (dilution of Ab varies).
• Wash 3 x 5 min. in PBS.
• Incubate with secondary antibody conjugated to a fluorescent label (e.g. FITC, ALEXA, TRITC etc) for 1 hour at room temp.
• Wash with PBS 3 x 5 min.
• Countersatin with DAPI if desired.
• Wash with PBS 3 x 5 min.
• Coverslip with anti-fade mounting medium.