Immunocytochemistry of Skin Fibroblasts

Skin fibroblasts cell lines from the IP and control (C1, C2) were counted and seeded on coverslips (1.2 × 10⁶ cells per 12 mm coverslip) by overnight incubation in 12-well plates [25 (link)]. Afterwards, cells were fixed for 20 min in 4% paraformaldehyde (PFA, Carl Roth, Karlsruhe, Germany) and blocked for 30 min in phosphate-buffered saline (PBS, Chemsolute, Renningen, Germany) with 0.1% Tween 20 (AppliChem, Darmstadt, Germany) and 5% bovine serum albumin (BSA, fraction V, Carl Roth). The ICC staining was performed with primary Anti-RBM10/S1-1 antibody (Abcam, Cambridge, UK, ab72423, 1:300) at room temperature for 2 h. Later, skin fibroblasts were incubated for 2 h at room temperature with secondary antibodies Alexa Fluor 568 (Thermo Fisher Scientific, A-10037, 1:2000). Each incubation was terminated by washing the cells in PBS supplemented with 0.2% Tween 20. Coverslips were mounted with DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL, USA) and slides were analyzed with a fluorescence microscope (Axio Observer, Zeiss, Oberkochen, Germany) and Axiocam 512 mono (4248 × 2832 pixles). Microscope images were examined with ImageJ software (ImageJ, Bethesda, Maryland, USA).