Ab16113

The human hair follicloids were first blocked in PBS containing 3% goat serum (Abcam, Cambridge, CB2 0AX, UK) and 0.3% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) for 1 h at 25 °C and subsequently incubated overnight with anti-cytokeratin 14 SP53 (ab119695; Abcam), anti-K40 AE13 (ab16113; Abcam), and anti-versican (ab19345, Abcam) antibodies at 4 °C. The samples were incubated with the corresponding secondary antibodies (Alexa Fluor® 488 ab150077; Abcam) suspended in the blocking solution for 2 h at 25 °C and finally stained with 4′,6-diamidino-2-phenylindole in PBS for 10 min. Next, 10-μm-thick frozen sections were prepared using the same steps as histological staining. An all-in-one fluorescence microscope (BZ-X810, Keyence) was used for fluorescence imaging.

Immunofluorescence was performed as described previously [29 (link)]. Briefly, the skin samples were isolated from mice (30 days), and fixed using 4% paraformaldehyde, followed by embedding in paraffin and dissection into 5-μm sections. Paraffin-embedded sections were microwave pretreated, and incubated with corresponding primary and secondary antibodies (Invitrogen), followed by DAPI staining. The corresponding antibodies were as follows: bromodeoxyuridine (BrdU; ab8152, mouse, 1 : 200, Abcam, Cambridge, UK), cleaved caspase-3 (ab13847, rabbit, 1 : 100, Abcam), GATA binding protein 3 (Gata-3; #5852, rabbit, 1 : 1600, Cell Signaling Technology, Beverly, MA, USA), lymphoid enhancer factor1 (Lef1; #2230, rabbit, 1 : 200, Cell Signaling Technology), Ki67 (#12075, rabbit, 1 : 50, Cell Signaling Technology), and alpha-esterase 13 (AE13; ab16113, mouse, 1 : 100, Abcam).
Immunofluorescence for HFSCs: HFSCs were placed on poly-d-lysine-coated coverslips, fixed using 4% paraformaldehyde for 20 min, and then permeabilized using 0.1% Triton X-100/PBS for 3 min. The HFSCs were then incubated with the following specific primary antibodies: Lef1 (#2230, rabbit, 1 : 200, Cell Signaling Technology), K19 (#12434, rabbit, 1 : 50, Cell Signaling Technology), CD200 (sc-71762, mouse, 1 : 100, Santa Cruz, Santa Cruz, CA, USA), and β1-integrin (ab95623, Rat, 1 : 500, Abcam).