Rabbit anti c fos polyclonal antibody

The rabbit anti-c-fos polyclonal antibody, rabbit anti-caspase-3 polyclonal antibody, rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) polyclonal antibody, and rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody were purchased from Abcam (Abcam, USA). The monoclonal mouse anti-claudin-5 antibody was purchased from Invitrogen (Invitrogen, USA). The monoclonal mouse anti-matrix metalloproteinase-2 (MMP-2), monoclonal mouse anti-MMP-9, and monoclonal rabbit anti-occludin antibodies were purchased from Abcam (Abcam, USA). RIPA buffer and the BCA kit were purchased from Beyotime (Shanghai, China). The mouse IL-6 ELISA kit and the IL-1β ELISA kit were obtained from R&D Systems (Minneapolis, MN, USA).

Sections (40 µm) containing the CeA were washed 3 × 10 min in PBS and incubated in blocking buffer (2.5% normal goat serum with 0.3% Triton X-100) for 2 hr at RT. Subsequently, sections were incubated in rabbit anti-c-Fos polyclonal antibody (1:1000 in blocking buffer; catalog no. 190289, Abcam, Cambridge, United Kingdom) for 48 hr at 4 °C. Sections were then washed 3 × 10 min in PBS and incubated in goat anti-rabbit Alexa Fluor 647 (1:500 in blocking buffer; catalog no. A32733, Invitrogen, Carlsbad, CA) for 2 hr at RT. After 3 × 10 min washes in PBS, sections were mounted on microscope slides and coverslipped with Prolong Gold Antifade Reagent with DAPI (Invitrogen, catalog no. P36935). Sections were imaged using a Keyence (Osaka, Japan) BZ-X800 fluorescent microscope at ×20 magnification and Fos⁺ cells were quantified manually by a blinded experimenter. Four sections representative of the CeA anterior-posterior axis (~bregma –1.8––2.8) from each animal were used for analysis.