Histochemical and Immunohistochemical Detection of lacZPositive Cells at the Light Microscopic Level

The injected embryos can be examined for the presence and distribution of ZacZ-positive cells at a later embryonic age or at some perinatal or postnatal time point. Since it is beyond the scope of this chapter to describe individual perfusion protocols for animals of every developmental age, a protocol that can be followed for adult animals will be given in the greatest detail.

The total number of pups should be counted after the pregnant dam has given birth to the injected embryos. Often, fewer pups are born than the number of embryos that were injected. Moreover, one can not correlate individual pups with the number they were assigned prenatally at the time of surgery unless the injection site was substantially different (i.e., right hemisphere vs left hemisphere, or big differences in the volume of retrovirus injected). If the injected animals are retrieved at a later embryonic time point, individual injected animals can be distinguished by their position along each uterine horn.

3.7.1.1. Perfusion

1. Anesthetize the experimental animal by placing it in a chamber lined with gauze that has been treated with ether.

2. Extend and immobilize the limbs of the fully anesthetized animal and follow standard intracardiac perfusion procedures. Make a longitudinal incision over the sternum from neck to xiphoid process (see Note 42).

3. Lift the skin and fur over the xiphoid process and cut laterally with scissors several centimeters in both directions.

4. Grasp the xiphoid process with toothed forceps or a hemostat and then free the lower margins of the ribs from any attachments to the abdominal organs. Carefully snip the diaphragm at its midline attachment to the sternum. Free the diaphragm from the lower margin of the rib cage.

5. Free the rib cage by cutting along the axillary line on each side; begin at the lateral margins of the abdominal incision. Make sure the mediastinal structures underlying the rib cage are detached from the ribs.

6. Retract the severed rib cage; be careful not to put any tension or pressure on the structures in the neck which will interfere with the flow of fixative to the brain (see Note 43).

7. In rapid succession, snip the right atrium so the animal begins to exsanguinate and insert a cannula or syringe needle, connected to tubing of the peristaltic pump, into the left ventricle (see Note 44).

8. Turn on the peristaltic pump and perfuse the anesthetized rodent with 4% paraformaldehyde, 0.2% glutaraldehyde in 0.1 M phosphate buffer (see Note 45).

9. After completion of the perfusion, remove the brain from the skull and transfer it to a container with fixative for not more than 1 h (see Note 46).

10. Section the brain or part of brain of interest on a Vibratome at a chosen thickness (e.g., usually 50-100 pm). Collect the sections in 0.1 M phosphate buffer in a 24-well culture dish or some other partitioned tray.

3.7.1.2. For Histochemical Detection of lacZ

1. Incubate the sections overnight at room temperature in staining buffer containing 1 mg/mL X-gal, 0.02% sodium deoxycholate and 0.01% Nonidet P-40 (NP-40)

2. Rinse the sections in 0.1 M phosphate buffer and mount on Superfrost slides for examination. Alternatively, the sections can be processed for the ultrastructural visualization of the lacZhistochemical reaction product (see Subheading 3.7.2.). Fig. 2A demonstrates the appearance of two histochemically stained lacZ-posi-tive cells.

3.7.1.3. For Immunohistochemical Detection of lacZ (After Collection of the Sections in 0.1 M Phosphate Buffer)

1. Incubate the sections in blocking serum for 1-2 h at room temperature.

2. Incubate the sections in the anti-P-galactosidase antibody diluted in blocking serum overnight on a rotator at 4°C.

3. Rinse the sections three times in PBS.

4. Incubate the sections in the secondary antibody conjugated to the desired fluoro-chrome (see Note 47) and proceed with standard immunohistochemical procedures. Fig. 2B demonstrates the appearance of an immunohistochemically stained lacZ-positive cell.

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