Rat anti gfap antibody

Avidin–biotin complex (ABC) technique was used for immunohistochemical detection of glial fibrillary acidic protein (GFAP) within the ACC. Dewaxed and dehydrated sections were incubated overnight at 4 °C with rat anti-GFAP antibody (Thermo Fischer Scientific, Cat#13–0300; 1:200). Visualization was done using commercial ABC (Santa Cruz Biotech, CA, USA) for 30 min. Afterwards, sections were exposed to 3,3′-diaminobenzidine tetrahydrochloride (DAB) as the chromogen and then counterstained with hematoxylin. Positive GFAP reaction was detected as the brown coloration of the astrocytes including their bodies and processes and GFAP percentage of immunoreactivity was recorded.

Immunohistochemical detection of glial fibrillary acidic protein (GFAP) within the striatum was carried out using the avidin–biotin complex (ABC) method. Sections were dewaxed, dehydrated, and incubated with the rat anti-GFAP antibody purchased from Thermo Fischer Scientific (Cat#13–0300; 1:200). Afterward, PBS-washed sections were incubated with the secondary antibody HRP EnVision kit (Dako, CA, United States). The bound antibody was visualized using the commercial ABC (Santa Cruz Biotech, CA, United States) system with chromogen 3,3′-diaminobenzidine (DAB) tetrahydrochloride, followed by hematoxylin as counter stain. The GFAP-positive reaction was observed by brown coloration of astrocytes, including their bodies, and processes and the GFAP percentage of immunoreactivity were calculated from the average of six non-overlapping fields.

Immunohistochemically examinations were conducted using the same brain sections used for autoradiography with [¹¹C]DAC. The sections were soaked in hexane for 10 min at room temperature, subsequently fixed with 4% paraformaldehyde, and then washed with phosphate-buffered saline. Respective primary antibody incubations were performed using a rabbit anti-mouse TSPO antibody (NP155, 1:1000)^{24 (link)}, a monoclonal mouse anti-rat CD11b antibody (1:100, AbD Serotec) and a rat anti-GFAP antibody (1:500, Thermo Fisher Scientific). The sections were incubated with the primary antibodies overnight at 4 °C. After the first immunoreaction, the sections were incubated with Alexa 647-conjugated anti-mouse IgG secondary antibody (1:500; Thermo Fisher Scientific), Alexa 546-conjugated anti-rat IgG antibody (1:500; Thermo Fisher Scientific) and biotin-conjugated anti-rabbit IgG secondary antibody (1:1000; Thermo Fisher Scientific) for 1 h at room temperature, followed by tyramide signal amplification using a Fluorescein System (FITC) (PerkinElmer, Waltham). The sections were washed with phosphate-buffered saline and mounted with medium (Vector Laboratories). Fluorescent images were captured using a fluorescence microscope (BZ-9000, Keyence).