Immunohistochemical Analysis of Brain Samples

Immunohistochemical studies were performed as described previously25 (link). Coronal brain sections were incubated in xylene and passed through series of graded alcohols and then subjected to antigen retrieval using the antigen retrieval solution (Vector Lab, Inc. CA). Tissue sections were incubated in 3% H₂O₂ solution for 20 min and then subjected to blocking using the vector lab blocking kit. Tissue sections were incubated overnight with Ki-67 (1:100), Cleaved Caspase-3 (1:200) and Bcl-2 (1:50) and then with secondary antibodies for 45 min at room temperature. Immunoreactivity was visualized by using the DAB substrate and counterstained with haematoxylin (Vector Lab, Inc. CA). The proliferative index was calculated as percentage of Ki-67-positive cells in five randomly selected microscopic fields at 20X per slide. TUNEL analysis was performed using the In situ Cell Death Detection Kit (Roche, Indianapolis, IN) as per the manufacturer’s protocol, and five randomly selected microscopic fields in each group were used to calculate the relative ratio of TUNEL-positive cells. DAPI was used to visualize the nuclei.