Rats were anesthetized and sacrificed by perfusing through the heart with 4% paraformaldehyde in PBS (pH 7.4). Brains were removed, postfixed overnight and sectioned by the Department of Pathology, Tianjin Huanhu Hospital. The sections were pretreated with xylene and then incubated with primary antibody against rat Fas ligand (Abcam Anti-Fas Ligand antibody, ab15285), at 4 °C overnight. Next, sections were incubated with complement, HRP conjugate, and DAB, orderly (Abcam EXPOSE Mouse and Rabbit Specific HRP/DAB Detection IHC kit, ab80436). Next, sections were counterstained with hematoxylin. Images were captured by a microscopy (Olympus).
The total FasL immunostaining score was computed according to Wang Y et al.43 (link). Briefly, the percentage of positive was defined as 0 (<5%, negative), 1 (5–25%, sporadic), 2 (25–50%, focal), or 3 (>50%, diffuse). Staining intensity was defined as 0 (no staining), 1 (weak staining), 2 (moderate staining), or 3 (strong staining). The total score of immunostaining was computed as the percentage positive score × staining intensity score, and ranged from 0 to 9. We examined five successive fields as a section and summed the scores.
The total FasL immunostaining score was computed according to Wang Y et al.43 (link). Briefly, the percentage of positive was defined as 0 (<5%, negative), 1 (5–25%, sporadic), 2 (25–50%, focal), or 3 (>50%, diffuse). Staining intensity was defined as 0 (no staining), 1 (weak staining), 2 (moderate staining), or 3 (strong staining). The total score of immunostaining was computed as the percentage positive score × staining intensity score, and ranged from 0 to 9. We examined five successive fields as a section and summed the scores.