Vectorshield hardset mounting medium with dapi

First, deparaffinisation and antigen retrieval were performed on 6 µm back skin sections using the Trilogy pretreatment solution (Cell Marque). Then, the sections were incubated in 10% donkey serum (Abcam) diluted in PBS containing 0.3% Triton X-100, at room temperature for 1 hour. Subsequently, the sections were incubated with primary antibody against BrdU (rat, Abcam) or GFP (rabbit, Santa Cruz) at 4 °C overnight. After washing 3 times with PBS containing 0.1% Tween-20, the sections were incubated with the appropriate Alexa 488/555 conjugated secondary antibody (Molecular Probes) for 1 h at room temperature. After washing 3 times with PBS containing 0.1% Tween-20, the samples were finally mounted in Vectorshield hardset mounting medium with DAPI (Vector Laboratories). Lgr5/6GFP and Krt14 expression in primary SCC cryosections was visualised by incubation with anti-GFP (chicken, Abcam) and anti-Krt14 (rabbit, Covance) antibodies, respectively.