Immunofluorescent Analysis of Rat Brain Markers

As previously described [40 (link), 41 (link)], rats (n = 8 per group) were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and perfused with PBS followed by fresh 2% glutaraldehyde and 4% paraformaldehyde through the ascending aorta. The brain was collected and fixed in 4% paraformaldehyde for 4 h and then dehydrated in 30% sucrose overnight at 4°C. The brain tissues were embedded in the optimal cutting temperature compound and finally cut to a thickness of 15 μm in a -20°C cryostat for immunofluorescent staining. After blocking in 10% normal goat serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the tissue sections were incubated overnight at 4°C in 10% normal goat serum in PBS containing primary antibodies such as rabbit anti-pCREB, 1 : 150, Cell Signaling Technology; rabbit anti-BDNF, 1 : 100, Abcam; mouse anti-Neun, 1 : 300, Abcam; mouse anti-GFAP, 1 : 500, Abcam; and mouse anti-Iba1, 1 : 500, Abcam. Afterwards, these slices were incubated with Alexa Fluor 594-conjugated goat anti-rabbit secondary antibodies and Alexa Fluor 488-conjugated goat anti-mouse secondary antibodies (1 : 600, Jackson ImmunoResearch) for 1 h. Then, they were washed in PBS and cover-slipped with VECTASHIELD Mounting Medium with DAPI (Cat: H-1200, Vector Lab). Images were captured by a microscopic imaging system (Olympus BX61 and FluoView).