The pretreatment of the slides consists of washing in isotonic buffer, blocking the endogenous peroxidase activity with H202, and blocking nonspecific binding with BSA and serum. The primary antibody is incubated overnight at 4°C.
2. Treat with 3% (v/v) H202 in PBS containing 0.1 % (w/v) Na Azide for 3 min.
3. Wash in PBS containing 0.1% (w/v) saponin, 1% (w/v) BSA, and 2% (v/v) blocking serum for 10 min.
4. Drain slides and pipet on 50-100 |JL of first (primary) antibody appropriately diluted (see Note 2) in PBS containing 0.1 % (w/v) saponin and 1% (w/v) BSA. Incubate overnight at 4°C in a moist chamber.
After a thorough wash, apply the secondary antibody and incubate 1-2 h.
5. Wash twice in PBS containing 0.1% (w/v) saponin for 5 min each.
6. Wash in PBS containing 0.1% (w/v) saponin, 1% (w/v) BSA, and 2% (v/v) serum for 10 min.
7. Drain slide and pipet on 50-100 mL of secondary antibody appropriately diluted (see Note 2) in PBS containing 0.1% (w/v) saponin and 1% (w/v) BSA. Incubate in a moist chamber for 1-2 h.
Following a second thorough washing, apply the streptavidin-per-oxidase complex to the sections and incubate up to 1 h.
8. Wash twice in PBS containing 0.1% (w/v) saponin for 5 min each.
10. Drain the slide and pipet on 50-100 mL of streptavidin peroxidase complex diluted 1:200 in PBS. Incubate in a moist chamber for 45-60 min.
Following a thorough wash, apply the chromogen DAB (see Note 1) to the sections to react with the bound peroxidase enzyme, which produces an insoluble dark red-brown reaction product. The background and negative control sections should be light tan.
12. React with 0.05% (w/v) DAB containing 0.01% (v/v) H202in PBS. The solutions used should be mixed just before use and protected from bright light. Incubate for 5 min.
13. Stop the reaction by rinsing in PBS for 5 min.
14. Post fix in 1% (v/v) glutaraldehyde (see Note 5) for 10 min.
Lightly counterstain (see Note 6) with neutral red (especially good for brain or spinal cord sections), or use Hematoxylin or Hematoxylin eosin (see Chapter 45). Some slides should be kept with no counterstain.
16. Stain in neutral red for 2-4 min., dehydrate in each of ethanol 70, 95, 100, and 100% (v/v) for 15 s each. Clear in two changes of fresh xylene, 2 min for each wash.
17. Cover slip using a xylene-based mounting media.
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