Anti c fos antibody

As previously described (Han et al., 2020 (link)), mice were anesthetized and perfused transcardially with 4% paraformaldehyde (PFA) in phosphate buffer saline (PBS), and tissues were fixed in 4% PFA at 4°C for 12 h. After dehydration by 30% sucrose, brain tissue was cut into 30-μm-thick sections using a freezing microtome (Leica CM30505, Germany). The posterior portion of the BLA-containing sections was permeabilized with 0.3% Triton X-100 and 5% donkey serum in PBS for 2 h and then incubated with rabbit polyclonal anti-c-Fos antibody (1:1,000, Abcam, Cambridge, United Kingdom, ab190289) or rabbit polyclonal anti-PV antibody (1:1,000, Abcam, ab11427) at 4°C overnight. After washing three times with PBS, sections were incubated with Cy3-labeled anti-rabbit secondary antibody (1:1,000, Absin, Shanghai, China, abs20024) for 1.5 h at room temperature and then given three 10-min washes in PBS. Afterward, sections were incubated with DAPI solution (1:5 for 15 min, Beyotime, Shanghai, China, C1005) for nuclear labeling and coverslipped with anti-fade mounting medium. Images were captured using an EVOS fluorescence microscope (Invitrogen, United States). For c-Fos immunostaining, mice subjected to several behavioral tests were perfused 1.5 h after the tests.

The cells were harvested and homogenized in RIPA lysis buffer (Sigma-Aldrich). The concentrations of the protein samples were determined by the Bradford method. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 10% gel) and transferred to membranes. After blocking with 5% skim milk for 1 h, the membrane was incubated with anti-P-gp antibody (Abcam), anti-c-fos antibody (Abcam) and anti-actin antibody (Sigma-Aldrich) at 4 °C overnight. The next day, the membranes were washed three times with Tris-buffered Saline-Tween 20 (TBST), and incubated with the secondary antibodies (Proteintech Group) for 1 h. After washing with TBST, the target proteins were visualized and subjected to image analysis.