Immunostaining for lacZ

1. Dissect embryos in 4% paraformaldehyde (in Ca2+ and Mg2+-free PB) and fix for 4 h.

2. Wash the embryos in 0.2 M sucrose (1 h) and then immerse in OTC briefly and immediately before freezing slowly in liquid nitrogen vapor, followed by immersion in liquid nitrogen.

3. Cut 60-pm thick sections using a cryostat and store at 4°C until required, then go to Subheading 3.6.3., step 1.

3.6.2. Adult Mice and Body Organs

1. Anesthetize adult mice with 2.5% avertin (0.017 mL/g of body wt) and perfuse via the left ventricle with 0.5% w/v sodium nitrate in half-strength phosphate-buffered saline (PBS).

2. Dissect the required organ, and postfix with the same sodium nitrate fixative (2 h).

3. Section the tissue with a vibratome at 50-100 pm thickness.

4. Store sections for immunohistochemistry in PBS/0.02% sodium azide at 4°C until required. Then go to Subheading 3.6.3., step 1.

3.6.3. Detection

1. Wash sections from Subheadings 3.6.1. and 3.6.2. selected for detection of P-galactosidase in PBS/10% normal horse serum /0.02% Triton X-100 at room temperature (12 h).

2. Incubate sections with E. coli P-galactosidase antibody (diluted 1:1000 in PBS/ 10% normal horse serum/0.02% Triton X-100) overnight at room temperature.

3. Wash sections in PBS (3 x 10 min), and then incubate with 2° antibodies. either biotinylated goat antirabbit IgG (diluted 1:400 in 0.02% Triton X-100 in PBS) or Texas red donkey antirabbit IgG (diluted 1:500 in 0.02% triton X-100 in PBS) for 2 h.

4. Remove unbound antibody by washing in PBS (10 min).

5. If using biotinylated goat anti-rabbit IgG, incubate the sections in streptavidin-peroxidase complex (diluted 1:100 in 0.2% Triton X-100) for 2 h.

6. Visualize the immunochemical reaction by washing sections with diaminobenzidine (0.5 mg/mL) in the presence of 0.01% H2O2 until the brown color is apparent.

7. If Texas red-conjugated donkey antirabbit IgG is used, then incubate the sections in streptavidin-FITC (diluted to 1:100 in 0.02% Triton X-100 in PBS) for 2 h.

8. Wash sections in PBS (3 x 10 min), and view either by light or fluorescence microscopy. Sections may be counterstained with 0.1% nuclear fast red.

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