Multi beads shocker

To measure the CD31 and F4/80 protein levels, mice were sacrificed and wound tissues were harvested 7 days after treatment. Briefly, the harvested wound tissues were homogenized using Multi-beads shocker^® and added to the T-PER Reagent (Thermo Fisher Scientific) consisting of proteinase and dephosphorylation inhibitors (Thermo Fisher Scientific). Total protein lysates were obtained after the elimination of debris by filtering with 0.22 μm filters (Millipore). Total protein (10 μg) was separated using a mini-protean TGX Stain-Free^TM gel (BioRad), and then transferred to PVDF membranes using the Trans-Blot^® Turbo^TM transfer system (BioRad). After blocking with the PVDF Blocking Reagent Can Get Signal^® (TOYOBO) for 1 hour at room temperature, the membranes were incubated with primary antibodies against CD31, F4/80, or tubulin (Sigma-Aldrich) at 4 °C overnight, followed by the appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour. Expression was visualized by chemiluminescence with a digital luminescent image analyzer (LAS-4000, GE Healthcare).

To evaluate the inflammatory response, ELISA kits (R&D Systems) were used to detect the contents of transforming growth factor β1 (TGF-β1) and interleukin-1beta (IL-1β) in lung tissue lysates as previously described [25 (link)]. Briefly, the lung tissues were homogenized using Multi-beads shocker® and added to the T-PER reagent (Thermo Fisher Scientific) consisting of proteinase and dephosphorylation inhibitors (Thermo Fisher Scientific). Lung tissue lysates (100 µg protein) were added to each well and measured following the manufacturer’s instructions. The optical density was measured at 450 nm using a microplate reader (Multiskan Fc, Thermo Fisher Scientific).