3.3.1. Quick Method for Abundant Antigens
Following dewaxing in a descending series of alcohols (100 to 50% v/v) and equilibration in PBS, the following routine protocols are recommended. All incubations are carried out in an humidified chamber to prevent drying out at any stage during immunostaining.
1. Incubate for 2 h in primary antibody, diluted in 0.1% (w/v) BSA/PBS, at room temperature.
3. Incubate for 1 h in species-specific anti-IgG antibody conjugated to FITC, and diluted in 0.1% (w/v) BSA/PBS (1 in 100 recommended).
5. Mount in glycerol-based antiquench mountant, and seal cover slip with nail varnish.
3.3.2. Amplification Method for Less Abundant Antigens
1. Preblock in 1% (v/v) normal serum (from the species in which the intended secondary antibody is raised), 0.1% (w/v) BSA in PBS for 30 min.
2. Incubate overnight at 4°C in primary antibody, diluted in the blocking solution.
4. Incubate for 1-2 h at room temperature in biotinylated secondary antibody diluted in the blocking solution (1 in 200 recommended).
6. Incubate for 1 h at room temperature in Extravidin-FITC diluted in PBS (1 in 100 recommended).
8. Mount in glycerol-based antiquench mountant, and seal cover slip with nail varnish (see Notes 7-10).
3.3.3. Immunoperoxidase Method for Less Abundant Antigen
1. Follow steps 1-5 in Subheading 3.3.2.
2. Incubate for 1 h at room temperature in Vectastain Elite ABC reagent (prepared 30 min before use).
4. Incubate with appropriate substrate, either DAB (brown) or Vector SG (black) or Vector VIP (deep purple).
5. Wash in distilled water (5 min).
6. Counterstain, dehydrate in IMS, clear in xylene, and mount in DPX. 4. Notes
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