Detection of lacZ Positive Cells at the Light Microscopic Level

1. Perfusion instruments:

a. Cannula for directing the flow of fixative into the left ventricle.

b. 2 Hemostats.

c. Scissors.

d. Small toothed forceps.

e. # 10 Scalpel blade holder.

2. Peristaltic pump for delivery of the fixative (Masterflex Pump #7520-24, Cole-Parmer Instrument Co., Vernon Hills, IL).

3. Ether chamber: Any container with a lid large enough to accommodate the experimental animal during the anesthetization procedure and resistant to ether fumes is suitable.

4. Fixative: 4% paraformaldehyde, 0.2% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2 prepared as in Subheading 2.1.3., item 2b.

5. Histochemical detection of lacZ-positive cells: In addition to the chemicals listed in Subheading 2.1.3., item 2:

a. Sodium deoxycholate.

c. 24-Well tissue culture plates.

d. Vibratome (see Note 10).

e. Superfrost slides (see Note 11, #12-550-15, Fisher Scientific, Pittsburgh, PA).

6. Immunohistochemical detection of lacZ-positive cells:

a. Blocking serum: 10% normal goat serum, 0.2% Triton X-100 in PBS, pH 7.4.

b. Primary antibody: mouse anti-P-galactosidase (Five Prime-Three Prime, Inc., CO), diluted 1:500 in blocking serum.

c. Secondary antibody: Anti-mouse IgG conjugated to fluorophore, diluted in blocking serum according to manufacturer's recommendations.

7. Detection of the phenotype of lacZ-positive cells: The investigator can double- or triple- label tissue sections to detect the expression of lacZ immunohistochemically, in conjunction with other cell-type specific antibodies of choice (14) (see Note 12).

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