Fitc anti alpha smooth muscle actin

Tissue sections were fixed with 4% paraformaldehyde (PFA) for 10 min, followed by permeabilization with 0.1% Triton X-100 for 10 min at room temperature. Non-specific sites were blocked using 3% bovine serum albumin (BSA) for 1 h at room temperature. Tissue sections were incubated at 4 °C overnight in 0.3% BSA and 0.1% Tween 20 containing primary antibodies anti-phospho-MLKL Ser345 (1:1000, Cell Signaling Technology, Danvers, MA, USA, 37333) or anti-phospho-CaMKII Thr287 (1:100, generated and validated by Dr. Singer’s lab) [16 (link),17 (link)] and FITC Anti-alpha smooth muscle actin (1:500, Abcam, Waltham, MA, USA, ab8211). Normal rabbit IgG (Thermo Scientific, Waltham, MA, USA, 026102) was used for the negative controls. After washing three times in PBS, sections were incubated with Alexa Fluor 594-conjugated secondary antibodies (1:500, Thermo Scientific, Waltham, MA, USA) for 1 h at room temperature in the dark. DAPI was used to stain nuclei. Images were acquired with a Nikon A1RS confocal microscope system, and analyzed using ImageJ software.

Cells were digested in a 0.05% pancreatic enzyme solution (Thermo Fisher, 25300054) for 3 min, and neutralized with Dulbecco's modified Eagle’s medium (DMEM, Boster, PYG0073) containing 10% fetal bovine serum (FBS). Cells were stained with Zombie Fixable Viability (BioLegend, San Diego, USA) before fixation by Fixation/Permeabilization Diluent (Thermofisher, 00-5223-56). Cells were stained with FITC Anti-alpha smooth muscle Actin (Abcam, ab8211, 1:50 dilution).