Rabbit secondary antibody

Protein expression was detected after extraction of proteins in tissues and cells using radio immunoprecipitation assay buffer (Invitrogen). After being separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was transferred to a polyvinylidene fluoride membrane (Millipore Corp, Billerica, MA, United States). After being sealed with 5% skim milk for 2 h, the membrane was incubated with primary antibodies at 4°C overnight, followed by 2-h incubation with corresponding rabbit secondary antibody (1:4,000, Abcam, Cambridge, United States) at 37°C. Immunoreactive proteins were visualized using enhanced chemiluminescence (Millipore). The primary antibodies included α-SMA (1:1,000; Abcam), fibronectin (1:1,000; Abcam), collagen IV (1:1,000; Abcam), p53-upregulated modulator of apoptosis (PUMA, 1:1,000, #24633, CST), apoptotic protease activating factor 1 (Apaf1, 1:1,000, ab2001, Abcam), B-cell CLL/lymphoma 2 (Bcl-2, #3498, CST), TGF-β1 (1:1,000, #MA5-15065, Thermo Fisher), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1,000; Abcam).

Mice were anesthetized with sodium pentobarbital, and transcardially perfused with 20 mL of 4% paraformaldehyde. The brain was removed and immersed in the same fixative at 4°C for 12 h. The tissue was embedded in paraffin and cut with a microtome (CV 5030; Leica, Germany) into 4um sections. The sections were incubated in 10% bovine serum in PBS containing 0.1% Triton X-100 for 30 min. Anti-mouse GFAP primary antibody (1:500; Abcam) was added to the slices, incubating at 4°C overnight. After washing, the sections were added with a rabbit secondary antibody (1:1000; Abcam) conjugated to AlexaFluor 488 and kept in the dark at room temperature for 2 h. DAPI was added to stain the cell nuclei. Micrographs were taken with a fluorescence microscope.

After extracting the proteins in cells using RIPA buffer (Invitrogen), the protein concentration was detected. After separating the protein samples by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred to polyvinylidene fluoride membranes (Millipore, USA). Subsequently, after soaking for 2 h in 5% skim milk, the membranes were incubated with primary antibodies overnight at 4°C. Then, the membranes were coated with rabbit secondary antibody (1:4,000; Abcam, UK) for 2 h at 37°C. The protein bands were observed using enhanced chemiluminescence reagents (Millipore). The primary antibodies included E-cadherin (1:1,000; Abcam), vimentin (1:1,000; Abcam), α-smooth muscle actin (α-SMA) (1:1,000; Abcam), fibronectin (FN) (1:1,000; Abcam), collagen I (Col I) (1:1,000; Abcam), collagen IV (Col IV) (1:1,000; Abcam), BMP7 (1:1,000; Abcam), and GAPDH (1:1,000; Abcam).