Thirty-six rats were used in the immunocytochemical study. Rats were continuously EEG monitored and killed at different time points after SE for subsequent immunocytochemical and histologic analysis. Rats were killed at 1 day (n = 2), 1 week (n = 4), 4 weeks (n = 2), 6 weeks (n = 3), and between 3 and 5 months after SE (n = 17). The last group was subdivided into rats with a progressive seizure evolution (n = 10; approximately five to 10 seizures per day; van Vliet et al., 2004) and rats with a nonprogressive form of seizure evolution (n = 7; on average, approximately one to two seizures per week; van Vliet et al., 2004); rats that were stimulated but that did not develop SE (non-SE, n = 3) and control rats (n = 5) also were included.
Rats were disconnected from the recording apparatus and deeply anesthetized with PTB (Nembutal; intraperitoneally, 60 mg/kg). The animals were perfused through the ascending aorta with 300 ml of 0.37% Na2S solution and 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. Horizontal sections (40 μm) were cut on a sliding microtome and collected in 0.1 M phosphate buffer for immunocytochemistry. In a subset of rats (n = 4), sagittal sections (40 μm) were cut. Horizontal sections between 5,100 and 5,600 μm below cortex surface (midlevel) 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 + 0.4% bovine serum albumin (BSA). Sections were incubated with anti–horse spleen ferritin (ferritin; polyclonal rabbit; 1:2,000, no F6136; Sigma-Aldrich, Zwijndrecht, The Netherlands) or monoclonal anti-rat CD11b/c (OX42; monoclonal mouse; Pharmingen, San Diego, CA, U.S.A.; 1:100, as a marker for microglia) 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/mouse immunoglobulin (Ig) (Amersham Pharmacia Biotech, Roosendaal, The Netherlands), diluted 1:200 in PBS. Sections were washed in PBS (3 × 10 min) and incubated for 30 min with AB-mix (Vectastain ABC kit, Peroxidase Standard pk-4000; Vector Laboratories, Burlingame, CA, U.S.A.). After washing (3 × 10 min) in 0.05 M Tris-HCl, pH 7.9, the sections were stained with 3,3′-diaminobenzidine tetrahydrochloride (30 mg DAB; Sigma-Aldrich), 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 the primary antibody eliminated all specific immunoreactivity. The intensity of ferritin immunoreactive cells was estimated semiquantitatively in the hippocampus, parahippocampus, and piriform cortex. The immunoreactivity was classified as follows: (+), moderate; +, strong; and ++, very strong. The frequency of such cells 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.