Rabbit secondary antibodies

The plastic-embedded sections were incubated with primary antibodies, anti-α-SMA and anti-Von Willebrand factor (both from Abcam), followed by incubation with rabbit secondary antibodies (Jackson ImmunoResearch). The nuclei were counterstained with DAPI (Sigma).

Cells cultured on Millicell® EZ SLIDE (MILLIPORE) were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 1 min at room temperature. After blocking with BSA (Sangon Biotech), samples were incubated with the primary antibody (1:200) overnight at 4°C, and the secondary antibody–labeled with fluorescein (1:200)–for 1 h at room temperature. Finally, DAPI was used to counterstain nuclei and the sample images were captured using a confocal microscope. For immunofluorescent staining of living cells to locate membrane proteins, cells were rinsed with cold PBS, followed by incubation with primary and secondary antibodies (1:500) for 1 h at 4°C. Next, the cells were fixed with 4% paraformaldehyde and stained with DAPI. For immunohistochemistry, slides were deparaffinized in xylene, and then antigen retrieval was performed using a citrate antigen retrieval solution. Endogenous peroxidase activity was blocked through the addition of hydrogen peroxide. After blocking with 10% BSA at room temperature, slides were incubated with primary antibodies at 4°C overnight. Following 1 h incubation with HPR-conjugated secondary antibody (anti-mouse, 1:500, Jackson ImmunoResearch, 115–035–003 or rabbit secondary antibodies 1:500 Jackson ImmunoResearch, 111–035–003), slides were developed in DAB (CST, 8059) and counterstained with hematoxylin.

Cells were washed and lysed with RIPA buffer (WB3100, NCM, China) containing protease inhibitors cocktail (B14001, bimake) on ice for 10 min. Then, protein lysate followed centrifugation in 4 °C for 10 min and the supernatant was collected. Protein supernatant were prepared with 5 × SDS loading buffer (P1040, Solarbio) and denatured at 100 °C for 5 min. Appropriate protein of samples were separated by 4–20% Genshare PAGE gel electrophoresis and electroblotted into NC membranes on eBlot™ L1 Protein Transfer System (GenScript). The membranes were incubated in 5% non-fat powdered milk (Cat No. 36101; Yeasen, Shanghai, China) in TBST (TBS with 0.1% Tween20) for 1 h at room temperature, followed by incubation with primary antibodies against specific proteins overnight: β-actin (1:5,000, Yeasen, 30101ES50), HSP90 (1:1000, 13171-1-AP, Proteintech), and OPA1(1:1000, Abcam, ab42364). The primary antibodies were diluted in universal antibody diluent (WB500D, NCM, China). The membranes were washed thrice of 10 min each time and incubated with the HPR-conjugated goat anti-mouse (1:10,000, Jackson ImmunoResearch, 115-035-003) or rabbit secondary antibodies (1:10,000 Jackson ImmunoResearch, 111-035-003) for 1 h at room temperature. Enhanced chemiluminiscence (ECL) was performed using the ECL kit (WB012, share-bio, China), visualized by the Bio-Rad system.