The simpler procedure, which uses a single tagged secondary antibody, can often be used (8). Animals are best perfusion fixed with PAF (9; see Chapter 45).
The pretreatment includes washing only with isotonic buffer and per-meabilizing the cell membranes with Triton-X-100 to allow the antibody access to the antigen, and to block nonspecific sticking of the antigen with BSA and serum. Incubate with the primary antibody overnight at 4°C.
2. Wash in PBS, pH 7.4, with 0.1% (v/v) Triton-X-100 for 10 min.
3. Treat with PBS containing 0.1% (v/v) Triton-X-100 and 10% blocking serum for 10 min.
4. Drain the slides and apply 50-100 pL of the primary antibody diluted (see Note 2) in PBS containing 1% (v/v) Triton-X-100. Incubate at 4°C overnight in a moist chamber.
After a thorough wash and a second application of blocking serum, apply the secondary antibody.
5. Wash twice for 10 min each in PBS containing 0.1% (v/v) Triton-X-100.
6. Treat with PBS containing 0.1% (v/v) Triton-X-100 for 10 min.
7. Incubate for 45-60 min in secondary antibody diluted (see Note 2) in PBS with Triton-X-100.
After a thorough wash, cover slip the sections with an aqueous mounting media.
8. Wash in PBS, three changes for 5 min each.
9. Mount the cover slip with permafluor.
Examine the sections with a fluorescent microscope equipped with narrow band width, blue excitation wavelength filters (see Note 1).
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