Skin Tissue Immunostaining and SMLM Imaging

Paraffin skin tissue sections (8 μm) of the skin biopsies of day 28 post-irradiation were mounted on super-frosted slides and processed and immunostained as described in detail elsewhere [13 (link),29 (link)], with the exception that immunostaining was conducted in TCTG buffer (TRIS, 1% Na-Casein, 0.1% Tween20, 0.1% fish-gelatin, pH 7.3–7.4) to reduce background. The slides were incubated with the primary antibodies for 1 h at 37 °C in TCTG buffer followed by three 5 min washes in TCTG and incubation with the secondary antibodies for 1 h at 37 °C. The antibodies, their sources and the dilutions used are listed in Table 1. After incubation with the secondary antibodies, sections were washed (3 × 5 min) in TCTG at 37 °C. Slides were supplied with 18 μL Prolong Gold Mounting Medium (Life technologies, Thermo Fisher Scientific, Darmstadt, Germany) containing DAPI (4′,6-diamidino-2-phenylindole) as DNA/nuclear counterstain and covered with a 24 × 60 mm cover slip. Preparations were cured for 2 days at RT and subjected to SMLM as described previously [29 (link)]. Widefield images were recorded using the ISIS fluorescence imaging system (MetaSystems, Altlussheim, Germany) equipped with an Axioimager 2i and 63× lens (both Zeiss, Oberkochen, Germany).