Mouse monoclonal αsma

Primary rat HSCs were seeded directly after isolation in 12-well plates containing 18 mm glass coverslips. Cells on coverslips were washed, fixed (4% PFA, 10 min) and permeabilized (0.1% Triton X-100, 10 min) prior non-specific blocking (2% BSA, 30 min). After blocking, cell on coverslips were incubated for 1 h at RT with the primary antibodies goat polyclonal collagen-I (Southern Biotech, 1310-01, 1/200) and mouse monoclonal αSMA (Sigma Aldrich (Munich, Germany), A5228, 1/100), washed three times with blocking solution and incubated with secondary antibodies goat anti-rabbit Alexa Fluor 488 or rabbit anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific, 30 min, at RT). After secondary antibody incubation, coverslips were washed three times and mounted with Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, Gdynia, Poland). Coverslips were air-dried, sealed using nail polish, stored at 4 °C and covered from light until further use. Images were obtained in a Zeiss 410 inverted laser scan microscope (Leica Microsystems, Wetzlar, Germany) with 16× or 40× magnification objectives using immersion oil and processed using ImageJ software (public domain, developed at the National Institutes of Health).