Histochemical Staining for lacZ with XGal

3.5.1. Small Embryos (7.5-11.5 D Postcoitum [p.c.]) and Fragments of (12.5-18.5 D p.c.) Embryos

1. Fix small embryos or fragments of older embryos in 4% paraformaldehyde/0.2% glutaraldehyde for 5-15 min.

2. Wash the embryos in PBS (2 x 5 min), and stain overnight in X-gal staining solution at 37°C in the dark.

3. Wash the embryos in 70% (10 min) ethanol and dehydrate in 70, 80, and 90% ethanol (10 min each), and then 100% ethanol (3 x 10 min).

4. Clear the embryos in xylene or Histolene (3 x 10 min), and impregnate in paraffin wax (3 x 20 min) before embedding in fresh wax.

5. 5-10 pm sections are cut and counterstained with nuclear fast red (see Notes 5-9).

3.5.2. Adult Embryos and Organs

1. Anesthetize adult animals and perfuse intracardially with 4% paraformaldehyde/ 0.2% glutaralde]hyde (or 2% paraformaldehyde) in 0.1 M phosphate buffer

2. Dissect the organ required and postfix in the same fxative for 1 h, followed by cryoprotection in 30% phosphate buffered 0.2 M sucrose solution overnight.

3. Cut frozen sections at 100-200 pm and briefly postfix with 4% paraformalde-hyde for 5 min to preserve the histology.

4. Rinse the sections in phosphate buffer (3 x 20 min), and then incubate in X-gal solution overnight at 37°C. in the dark.

5. Rinse the sections with PB (3 x 20 min) and examine under a dissecting microscope (see Note 10).

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