Histochemical Detection of lacZPositive Cells at the Ultrastructural Level

1. Day 1: As described in Subheading 3.7.1.1., perfuse the experimental animals, and remove and fix the brains. Collect 100 |im tissue sections and carry out the X-gal reaction.

2. Day 2: Aspirate the X-gal reaction mixture from each well, rinse the sections with 0.1 M phosphate buffer and post-fix the tissue sections for a few hours in 4% paraformaldehyde/2% glutaraldehyde in 0.1 M phosphate buffer.

3. Rinse the tissue sections three times in 0.1 M phosphate buffer, incubating for 5 min each time

4. Transfer the sections from each well of the 24-well plate to a fresh well of a 6-well tissue culture plate (see Note 48).

5. Aspirate the phosphate buffer and add 0.5 mL of 1% OsO4 in 0.1 M phosphate buffer (see Note 49) to each well (see Note 50). Place the 6-well plate containing the tissue sections on a rotator in the fume hood for 30 min.

6. Carefully remove the OsO4 to a toxic waste container and add the following solutions to counterstain (uranyl acetate) and dehydrate (ethyl alcohol) the tissue sections in each well for the stated times:

0.1 N sodium acetate 5 min, twice

1% aqueous uranyl acetate 30 min

0. 1 N sodium acetate 5 min

25% ethyl alcohol 2 min

50% ethyl alcohol 2 min

70% ethyl alcohol 10 min

95% ethyl alcohol 15 min, twice

100% ethyl alcohol 15 min, twice

7. Aspirate the alcohol from the wells and replace with a 1:1 mixture of 100% ethyl alcohol:Araldite resin. Leave overnight on a rotator at room temperature.

Fig. 2. Representative examples of the appearance of facZ-positive cells in the central nervous system at the light and electron microscopic levels. (A) Representative examples of histochemically stained facZ-positive neurons in the rat cerebral cortex resulting from an injection of retrovirus into the cerebral ventricles of the embryonic d 16 rat telencephalon. Note that the cell bodies and proximal dendrites are intensely stained (modified with permission from Luskin et al., 1993). (B) Representative example of an immunohistochemically stained neuron in the olfactory bulb resulting from a perinatal injection of retrovirus. The tacZ-positive cell was revealed using a primary antibody to P-galactosidase; diaminobenzidine was used as a chromogen to visualize the secondary antibody conjugated to horseradish peroxidase. In most instances immunohistochemistry can be used to label the fine processes of tacZ-positive cells (modified with permission from Luskin, 1993). (C) An electron micrograph showing a tacZ-positive (left) and unlabeled (right) neuron in the visual cortex from a rat that received an intraventricular injection of retrovirus at E16. The nucleus and nuclear membrane are conspicuously stained and in the cytoplasm the reaction product is associated preferentially with the endoplasmic reticulum, which extends into the apical dendrite (large arrows) and basal dendrite (arrowhead) of the labeled cell (modified with permission from Luskin et al., 1993).

8. Day 3: Remove the alcohol/Araldite mixture and replace with 100% Araldite. Change resin every 2 h so that the sections incubate in the resin at room temperature for at least 6 h.

9. Embed the tissue sections in Araldite resin as follows: Trim two acetate sheets for each slide. One piece should be the same size as the slide or slightly smaller and the other should be slightly larger.

10. Using a paper punch, make small holes in the corners of each piece of the smaller acetate sheets. These will become the bottom sheets of the microscope slide sandwich.

11. Place the lower acetate sheet on a microscope slide and place one small drop of plastic in each corner hole. These plastic drops will secure the acetate film to the slide. Place 6 small drops of embedding plastic on the acetate film (two rows of 3 drops evenly spaced) or one for each section that will be embedded. Carefully scoop the section out of the dish where it has been processed and place it on top of one of the drops of plastic. Cover each tissue section with one more small drop of embedding plastic.

12. Cover the tissue sections with a piece of the larger sheets of cut acetate film. Place weights or additional slides on top of each acetate sandwich to flatten the plastic during baking.

13. When ready to bake the tissue embedded between the acetate sheets, place them on cardboard and then place the cardboard on the oven rack for backing. This way the microscope slides will not stick to the metal oven rack if some of the plastic oozes out from between the slides.

15. The tissue is ready for trimming, cutting on an ultramicrotome and viewing with the electron microscope. Fig. 2C demonstrates the appearance of a lacZ-positive cell at the ultrastructural level.

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