Rats were disconnected from the recording apparatus and deeply anesthetized with pentobarbital (Nembutal; intraperitoneally, 60 mg/kg). The animals were perfused through the ascending aorta with 300 ml of 0.37% Na2S solution, 300 ml 4% paraformaldehyde, and 0.2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were postfixed in situ overnight at 4°C, dissected, and cryoprotected in 30% phosphate-buffered sucrose solution, pH 7.4. After overnight incubation at 4°C, the brains were frozen in isopentane (–25°C) and stored at –80°C until sectioning. The brains were cut on a sliding microtome and 40-μm horizontal sections were collected in 0.1 M phosphate buffer for immunocytochemistry. Rats were killed 1 day (n = 5), 1 week (n = 5), 4–6 weeks (n = 5), or 4–9 months (np-SE, n = 5; p-SE, n = 7; non-SE, n = 6) after the stimulation, including five sham-operated control rats (see also Table 1). Horizontal sections between 5,100 and 5,600 μm below cortex surface (midlevel) and 7,600 and 8,100 μm below cortex surface (ventral level) of the brain [according to (20)] of control and post-SE rats were stained with different immunocytochemical markers. For each animal, two sections were analyzed per level. Sections were washed in 0.05 M phosphate-buffered saline (PBS), pH 7.4, and incubated for 30 min in 0.3% hydrogen peroxide in PBS to inactivate endogenous peroxidase. Sections were then washed (2 × 10 min) in 0.05 M PBS, followed by washing (1 × 60 min) in PBS + 0.5% Triton X-100 + 0.4% bovine serum albumin (BSA). Sections were incubated with rabbit anti-MVP [1:100, a generous gift from Dr. L.H. Rome, described in (21)] in PBS + 0.1% Triton X-100 + 0.4% BSA at 4°C. Twenty-four hours after the incubation with the primary antibody, the sections were washed in PBS (3 × 10 min) and then incubated for 1.5 h in biotinylated sheep anti-rabbit or anti-mouse immunoglobulin (Ig; Amersham Pharmacia Biotech, Roosendaal, the Netherlands), diluted 1:200 in PBS + 0.1% Triton X-100 + 0.4% BSA. Sections were washed in PBS (3 × 10 min) and incubated for 1.5 h in streptavidin–horseradish peroxidase (Zymed Laboratories, San Francisco, CA, U.S.A.), diluted 1:200 in PBS + 0.1% Triton X-100 + 0.4% BSA. After washing in 0.05 M Tris-HCl, pH 7.9, the sections were stained with 3,3'-diaminobenzidin tetrahydrochloride (30 mg DAB; Sigma-Aldrich, Zwijndrecht, the Netherlands) and 750 μl 1% hydrogen peroxide in a 100-ml solution of Tris-HCl. The staining reaction was monitored under the microscope and stopped by washing the sections in Tris-HCl. After mounting on gelatine-coated slides, the sections were air dried, dehydrated in alcohol and xylene, and coverslipped with Entellan (Merck, Darmstadt, Germany). Omission of primary antisera, preincubation with excess of MVP protein, and incubation with normal goat serum eliminated all specific immunoreactivity. The intensity of MVP immunoreactive (IR) cells in microglia was estimated semiquantitatively in the hippocampus, parahippocampus, and piriform cortex. The immunoreactivity was classified as follows: (+), weak; +, moderate; and ++, strong. Frequency was classified as 1, sparse; 2, high; 3, very high; and 4, clusters formation. Sections were photographed by using bright-field illumination on an Olympus-Vanox microscope, equipped with a digital camera, and imported into Adobe Photoshop (version 7.0). This program was used to optimize contrast and brightness, but not to enhance or change the image content in any way.