*2.7. Immunocytochemistry*

After the isolation of primary murine hepatic stellate cells, the cells were cultured on coverslips for 24 h or 10 days. Cells were washed with PBS and fixed with formalin for 10 min. The cells were then washed three times with PBS and stored at 4 ◦C until further use. Prior to staining, the cells were permeabilized using PBS supplemented with 0.1% Triton-X (3 × 5 min). Afterwards, cells were incubated for 30 min with 0.1% Triton-X PBS containing 2% bovine serum albumin (BSA), to block non-specific binding sites. The cells were incubated overnight at 4 ◦C with anti-Desmin (1:200, RB-9014-P, ThermoFisher scientific), or anti-Vimentin (1:200, V5255, Sigma-Aldrich). Three wash-steps using 0.1% Triton-X PBS were applied, followed by incubation with donkey anti-rabbit Alexa Fluor 488 secondary antibody (1:200, A21206, ThermoFisher scientific), donkey anti-mouse Alexa Fluor 488 secondary antibody (1:200, A21202, ThermoFisher scientific), or Cy3-coupled mouse anti-αSMA primary antibody (1:100, C6198, Sigma-Aldrich) for 1.5 h. Coverslips were mounted with 4 ,6-diamidino-2-phenylindole (DAPI)-containing mounting medium (Dako, Denmark). Images were taken using the EVOS FL fluorescence microscope (ThermoFisher).
