Ab175

Primary astrocytes cultured in a 24-well plate were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde (Sigma, G5882-50 ML) for 15 min at room temperature after 13 DIV, and washed with 0.1 M phosphate-buffered saline (PBS) for 5 min. Samples were incubated with 0.1 M PBS containing 0.3% Triton X-100 (Sigma) and 10% donkey serum (Genetex, CA) for 1.5 h to prevent non-specific binding. Cells were incubated with a guinea-pig anti-GABA antibody (ab175; 1:1000; Millipore, MA) and a chicken anti-glial fibrillary acidic protein (GFAP) antibody (ab5541; 1:1000; Millipore) overnight at 4℃. After washing with 0.1 M PBS, cells were incubated with a DyLight 488- or 594-conjugated secondary antibody (1:500; Jackson Laboratory, ME) for 2 h at room temperature. After three rinses with 0.1 M PBS and staining with DAPI (1:3000; Thermo Scientific, MA), coverslips were mounted on Polysine microscopic glass slides (Thermo Scientific, MA). Images were acquired using a Nikon A1R confocal microscope.

Animals were deeply anesthetized using 2% avertin and perfused with 0.1M PBS, followed by 4% paraformaldehyde solution. Brains were post-fixed in 4% paraformaldehyde at 4℃ for 24 hrs and dehydrated in 30% sucrose at 4℃ for 48 hrs. Brains were then cut in coronal sections of 30 µm on a cryosection. Sections were blocked in 0.1 M PBS containing 0.3% Triton X-100 (Sigma) and 2% donkey serum (Genetex) for 1 hr at room temperature. Primary antibodies used are as following: chicken anti-GFAP (1:500, ab5541, Millipore), guinea pig anti-GABA (1:200, ab175, Millipore), chicken anti-proBDNF (1:200, ab9042, Millipore, epitope; fusion protein from mouse proBDNF containing only the prodomain region), rabbit anti-BDNF (1:50, sc20981, Santacruz, epitope; recombinant BDNF containing amino acid 130-247 from N-terminus). The brain samples with primary antibodies were incubated overnight at 4℃. Then, the sections were washed three times in 0.1 M PBS and incubated in proper secondary antibodies (1:500) from the Jackson Laboratory for 3 hrs. After three rinses in 0.1 M PBS and DAPI staining at 1:10000 (PIERCE), the sections were mounted on the Polysine microscopic slide glass (Thermo Scientific). Images were acquired using a Nikon A1R confocal microscope.

The primary antibodies used for immunostaining were as follows: chicken anti-GFAP (1:500; Millipore, ab5541), guinea pig anti-GABA (1:1000; Millipore, ab175), and rabbit anti-ABP1 (amiloride binding protein 1) (DAO) (1:500; Aviva Systems Biology, ARP41908_P050). Fluorescent secondary antibodies were purchased from Invitrogen or Jackson ImmunoResearch and used in 1:200 dilutions.