Normal serum

Acetone or PFA-fixed slides were washed in PBS and were blocked with 20% normal serum (rabbit or goat serum from Abcam) for 1 h. After washing steps in PBS, the slides were treated with primary antibodies for 1 h and were washed in PBS twice. The slides were then incubated with secondary antibody conjugated with AF488, AF568 or AF647 (10 μg/ml, ThermoFisher Scientific) for 1 h in the dark. The slides were then washed in PBS followed by dipping in deionised water briefly. The slides were then mounted with Fluoromount-G with DAPI (ThermoFisher Scientific).

Following deparaffinization through a series of graded alcohols, endogenous peroxidase activity was quenched in 3% H₂O₂ (in methanol; Carl Roth GmbH + Co. KG, Karlsruhe, Germany). Unspecific antigen-binding sites were blocked using 10% normal serum (in PBS; Abcam, Cambridge, UK), endogenous biotin was inhibited using the Avidin/Biotin Blocking kit (Vector Laboratories, Burlingame, CA, USA) followed by heat-induced epitope retrieval (0.01 M citrate buffer, pH 6.0; 800 W for 6 min). Sections were incubated overnight at 4 °C with antibodies directed against proliferating cell nuclear antigen (PCNA; Abcam), protein inhibitor of activated STAT1 (PIAS1; Abcam) or smooth muscle α-actin (SMA; Sigma). The next day, sections were incubated with secondary antibody (Molecular Probes Inc., Eugene, OR, USA), preformed avidin–biotin complexes (Vector Laboratories) and the peroxidase substrate 3-amino-9-ethylcarbazole (for PIAS and SMA) or 3, 3 -diaminobenzidine (for PCNA) (both Vector Laboratories) until color development. Sections were briefly counterstained with Gill’s hematoxyline (Sigma), mounted in ImmuMount (ThermoScientific, Waltham, MA, USA) and photographed (Olympus BX51 microscope, Olympus Europa SE & Co. KG, Hamburg, Germany).