2.9 Immunohistochemical staining
The brains were sectioned at 20 µm on a freezing
microtome after perfusion and fixation in 4%
paraformaldehyde (PFA). The frozen sections were blocked with 10%
donkey serum (v/v) for 1 hour and then incubated with GFAP antibody
(1:500, mouse monoclonal; Cell Signaling Technology; Cat# 3670, RRID:
AB_561049) at 4 °C for an additional 24 hours. GFAP staining was
visualized with Alexa 594-conjugated goat anti-mouse secondary antibody
(1:200, EarthOx, LLC, San Francisco, CA, USA) and Alexa 488-conjugated
goat anti-mouse secondary antibody (1:200, EarthOx). All images were
obtained by fluorescence microscopy (Leica DM2000, Leica, Wetzlar,
Germany) with Leica Application Suite-X software (Leica, Version 3.4.1).
The immunofluorescent intensity of the positive staining area was
included and optimized by a low and high threshold setup. To quantify
the number of GFAP-positive cells, the fields in the BLA area were
randomly selected under a confocal microscope at 20× magnification. The
related computer-assisted image analysis (ImageJ Software;
RRID:SCR_003070; Version 6.0) was conducted blindly by an investigator.