1. Fix embryos overnight in 4% w/v paraformaldehyde in PBS at 4°C (see Notes 1 and 2) using a large (20- to 50-fold by volume) excess of fixative.
3. Block endogenous peroxidase enzymes with PBS containing 0.05% (v/v) hydrogen peroxide, 1% (v/v) Triton X-100 at 4°C overnight on a rocker or slowly rotating wheel (see Note 3).
4. Wash in PBS containing 1% (v/v) Triton X-100 three times each for 1 h.
5. Dilute antibody (seeNote 4) in basal Eagle's medium containing 10% calf serum, 1% (v/v) Triton X-100 and 0.02% (w/v) sodium azide (see Note 5). Incubate for 2-4 d at 4°C with rocking.
6. Wash in PBS containing 1% v/v normal serum of same species as peroxidase-conjugated secondary antibody (e.g., normal goat serum for goat antimouse secondary antibodies) and 1% (v/v) Triton X-100, three times each for 1 h at 4°C.
7. Incubate with peroxidase-conjugated secondary antibody appropriate for the primary antibody used in step 5 (e.g., peroxidase-conjugated goat antimouse IgG or IgM for mouse MAbs). The secondary antibody is diluted in PBS containing 1% (v/v) Triton X-100 and 1% (v/v) normal serum as in step 6 at 4°C overnight. The precise dilution of secondary antibody should be determined empirically, but we generally find that dilutions of 1:100 are about right.
9. Wash in 0.1 M Tris-HCl, pH 7.2, twice for 30 min.
10. Incubate in 0.5 mg/mL DAB in 0.1 M Tris-HCl, pH 7.2, for 3 h at 4°C in the dark with rocking (see Note 6).
11. Replace with 0.5 mg/mL DAB in 0.1 M Tris-HCl, pH 7.2, containing 1 pL/mL hydrogen peroxide, and allow color to develop (usually 5-15 min; longer may be required if embryo is to be sectioned subsequently).
12. Reaction is stopped by several quick changes of tap water followed by several changes of PBS over a few hours.
13. Embryos can be further dissected at this point, and then cleared in 90% (v/v) glyc-erol, 1% PBS, 0.02% (w/v) sodium azide for storage or mounting for photography.
14. For sectioning, embryos are dehydrated through graded ethanols, equilibrated in xylene, and then embedded in wax as described in Chapter 41 (radioactive in situ hybridization to tissue sections, I.J.Mason) followed by sectioning at thicknesses of 7-15 pm.
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