Immunohistochemistry for Hippocampal Neurogenesis

We performed immunohistochemistry for visualizing glial fibrillary acidic protein (GFAP) positive astrocytes, BrdU+ newly born cells, and doublecortin (DCX) positive newly born neurons in the hippocampus. The methods employed for cutting thirty-micrometer thick coronal sections through the entire forebrain, collection, and storage of sections in cryobuffer, and immune-histochemical methods are described in our previous reports [15 (link), 46 (link), 52 (link), 53 (link)]. GFAP, BrdU, and DCX immunohistochemistry were done on every 15th or 20th section through the hippocampus's entire septotemporal axis in each animal. The primary antibodies comprised rabbit anti-GFAP (1:2000; Dako, Santa Clara, CA), mouse anti-BrdU (1:500; Abcam), and goat polyclonal anti-DCX (1:300; Santa Cruz, Dallas, TX). The secondary antibodies comprised anti-rabbit, anti-mouse, or anti-goat IgG’s (Vector Laboratories, Burlingame, CA). The avidin-biotin complex reagent and chromogen kits (vector gray or diaminobenzidine) were also purchased from Vector Labs. The sections were fixed on gelatin-coated slides, dehydrated, cleared, and coverslipped.