Ab6326

For organotypic co-culture, young adult (5 weeks old) C57BL/6 SCRM (BL6) mice were used. They were sacrificed by cervical dislocation and processed as previously described in (Marques-Torrejon et al., 2018 (link)). Samples were blocked in 10% normal goat serum and 0.2% Triton X-100 in PBS for 1 h and incubated for 48 h in blocking buffer with the appropriate primary antibodies: GFAP (1:500, Z0334 DAKO) KI67 (1:100 Thermo RM9106), SOX2 (1:100, AB5603, Millipore), NESTIN (1:10, Rat 401, Developmental Studies Hybridoma Bank), p53 (1:500, ab6326, Cell signalling), LRIG1 (1:100, R&D, AF3688), CD9 (1:100, 14–0091–82, eBioscience, 1:500), RFP (1:500, Abcam 62,341), BrdU (1; 1,000 Abcam, ab6326), SSEA1 (1:500, Biolegend, ab63260), Vimentin-40E (1:50, DSHB), CD133 (1:500, Milllipore, ab6326), CD9 (1; 500,14–0091–82, eBioscience), and pSMAD1/5 (1:500, Cell signalling, 9,516).

Tail whole-mount tissues were processed as previously described [12 (link)]. Primary antibodies were used at the following dilutions: rat anti-β4-integrin (CD104) (1:100, BD Biosciences, 553745), rabbit anti-caspase-3, active (cleaved) form (1:100, Millipore, AB3623), rat anti-BrdU (1:300, Abcam, ab6326), rat anti-Ki67 (1:100, eBioscience, 14-5698-82), rabbit anti-K14 (1:1000, BioLegend, 905304, polyclonal), mouse anti-K10 (1:100, BioLegend, 904301, or 1:100, Abcam, ab9026), guinea pig anti-K31 (1:100, PROGEN Biotechnik, GP-hHa1). All secondary antibodies (Alexa 488 or Alexa 546, Invitrogen) were used at 1:200 dilution. The mouse primary antibodies were blocked with MOM kit (Vector Laboratories). All samples were counterstained with Hoechst (Sigma, B2261) for 1 hour and mounted. To stain with anti-BrdU antibody, tail whole-mount pieces were blocked and incubated with 2N HCl at 37°C for 1 hour, further washed and stained. The stained whole-mount epidermis was observed by a confocal microscope (Zeiss LSM 700) with a ZEN 2010 software. All pictures were shown as projected Z-stack image, viewed from the basal side.