Immunohistochemistry

Briefly, detection of proliferating cells (“newborn” cells that have undergone mitogenesis) were marked by treating rats with the thymidine analog, bromodeoxyuridine (BrdU). Juvenile rats will be were treated with or without chronic CORT and BrdU. After varying treatment regimens, rat brains were fixed, sliced, and BrdU; and the neuronal marker protein, NeuN, was detected using fluorescent immunohistochemistry. Cell proliferation in the dentate gyrus was quantified using immunohistochemical detection of BrdU and counting BrdU-positive cells using epifluorescence microscopy. Neurogenesis (formation of newborn neurons) was assayed by immunohistochemical detection of colocalized BrdU and NeuN using confocal microscopy. Newborn cells and newborn neurons in the dentate gyrus of the hippocampus were identified by immunohistochemistry. Fluorescent antibodies allowed us to visualize cells containing BrdU and NeuN (a neuronal nuclear marker³¹). Brain slices were washed in PBS for 5 min, three times. The slices were then incubated in 1 M hydrochloric acid at 45°C for 1 h and washed six times with PBS and incubated in a blocking solution (PBS, 0.3% Triton X-100, 2% equine serum) for 1 h. Once blocking solution was removed, brain slices were placed in a 1:4000 solution of primary antibody (rat anti-BrdU; mouse anti-NeuN) in blocking solution. Slices were then incubated for 24 h at 4°C. Brain slices were then washed for five minutes in PBS three times and incubated with secondary antibodies (Alexa Fluor 594 donkey anti-rat, Invitrogen; Alexa Fluor 488 goat anti-mouse, Invitrogen) diluted at 1:1000 in PBS with 0.3% Triton-X 100 for 2 h. Slices were washed for 5 min in PBS three times and then mounted onto microscope slides.