Af761

For BrdU staining, antigen retrieval (DNA denaturation) was performed with 10mM sodium citrate (pH6) at 95 °C for 20 min, permeabilized in 0.1% Triton X-100 (T8787; Sigma) in PBS (PBST) for 3 × 10 min, then blocked with 20% goat serum (G6767; Sigma) in PBST for 30 to 60 min at room temperature. For P-cadherin staining only, 20% donkey serum (D9663; Sigma) was used instead of goat. Specimens were incubated with primary antibodies at 4 °C overnight. Primary antibodies were as follows: anti-RFP (1:500, 600-401-379; Rockland Immunochemical), goat anti-P-cadherin (1:200, AF761; R&D Systems), rabbit anti-non-muscle myosin IIB (1:200, 909901; BioLegend), and rat anti-BrdU (1:200, ab6326; Abcam). After six 1- to 2-h PBST washes, specimens were incubated at 4 °C overnight with Alexa Fluor–conjugated secondaries (Life Technology). Nuclei and F-actin were counterstained with DAPI (4′,6-diamidino-2-phenylindole; 1:5000, 62247; Thermo Fisher Scientific) and Alexa Fluor 488/635 Phalloidin (1:500, A12379, A34054; Invitrogen), respectively. Specimens were washed 6 times with PBST (1 to 2 h per wash) and then mounted on glass slides with 50% glycerol (356352; Calbiochem) in PBS. Z-stacks were acquired by a confocal microscope (TCS SP5; Leica) with oil immersion 40× and 63× objectives.

For histology analysis, Hematoxylin and Eosin (H&E) staining were performed according to standard protocols with minor modification: sections were incubated for 5 min in hematoxylin and 30 s in eosin solutions. Image acquisition was performed on a Leica DM4000 microscope. Only follicles with a clear and complete structure were chosen for length analysis. The length of hair follicles was measured by ImageJ.
For immunostaining, the frozen sections were blocked in PBS with 5% Donkey serum or 1% BSA and 0.25% Triton for 1–4 h at room temperature, then incubated with primary antibody at 4 °C overnight and were subsequently incubated with secondary antibodies conjugated with Alexa Fluor 488, 594 or 647 (1:1000, Invitrogen, California, USA). Nuclei were stained with DAPI (Solarbio, Beijing, China). The following primary antibodies were used: P-cadherin antibody (1:1000, AF761, R&D), K15 (1:100, ab52816, Abcam), and Lef1 (1:200, 2230, CST). Image acquisition was performed on a Zeiss microscope. For quantification, 5 sections from each sample were processed and 5 randomly selected areas of each section were quantified in Image J.