Integrin α6

At day 12 (late-anagen phase) after depilation, the back skin of control and K5-Cre;PDPN^flox/flox mice (n = 5 each) was dissected, minced using scissors, and digested in Dulbecco's Modified Eagle Medium supplemented with 2% fetal bovine serum (FBS), 1.2 mM CaCl₂, 3.5 mg/ml collagenase IV (Gibco, Grand Island, NY, USA), and 40 μg/ml DNase I for 20 min at 37°C under constant rotation. Samples were passed through a 70-μm cell strainer and washed with FACS buffer (PBS, 1% FBS, 2 mM ethylenediamine tetraacetic acid). After spinning down the cells (10 min, 1200 rpm), cell pellets were resuspended with FACS buffer and passed through a 40 μm cell strainer. Cells were stained with antibodies for 20 min. Antibodies were as follows: CD49f-APC (integrin α6, eBioscience, San Diego, CA, USA), CD34-FITC (BD Pharmingen, San Jose, CA, USA), CD31-PE (Biolegend, San Diego, CA, USA), and CD45-APC/Cy7 (Biolegend). For live/ dead discrimination, 7-AAD (Biolegend) was used. 7AAD^-, CD45^-, CD31^-, CD34⁺, integrin α6⁺ cells were considered HF stem cells and were sorted using a FACS Aria 2 instrument (BD Pharmingen).

Freshly prepared skin samples were embedded in OTC and frozen in liquid-nitrogen-cooled isopentane. Sections (7 μm) were then collected and stained with either hematoxylin and Eosin or Oil-Red O. Immunofluorescent and immunohistochemical staining of frozen sections was carried out following standard protocols. For immunohistochemical analysis, the signal was visualized in a DAB color developing solution and counterstained with hematoxylin when necessary. To detect BrdU-labelled cells, after permeabilisation and prior to incubation with the anti-BrdU antibody, sections were incubated for 20-30 minutes in 2 M HCl at 37 °C. The following antibodies were used: rabbit anti-human ASH1L (ab4477, Abcam, 1:50), keratin 1 (MK1, Covance, 1:1000), keratin 14 (MK14, Covance, 1:1000), Loricrin (Loricrin, Covance, 1:1000), Ki67 (Ki67, Novocastra, 1:1000), BrdU (ab6326, Abcam, 1:500) and CD49f (Integrin α6, BD Pharmingen, 1:200), goat anti-rabbit IgG-FITC (Sigma, 1:1000), and donkey anti-rat IgG-FITC (Sigma, 1:1000).

Mice skin tissue (dorsal and tail skin) at various postnatal day ages were collected and fixed by neutral buffered formalin (NBF) or directly embedded in OCT compound (Tissue-Tek) and frozen. Immunofluorescence assays (IF) and Immunohistochemical analysis (IHC-P) was performed as previously described^{7 (link)}. Paraffin embedded tissue blocks were sectioned and Haematoxylin and Eosin staining (H&E) was performed to analyze the phase of the hair follicle cycle. Immunofluorescence assays (IF) and Immunohistochemical analysis (IHC-P) was performed on OCT frozen tissue and Paraffin embedded block respectively²³ . Tail whole mount was performed as described previously^{48 (link)}. Briefly, tail skin was incubated in 5 mM EDTA followed by separation of epidermal sheet from dermis followed by fixing with 2% formaldehyde for 10 minutes. Nile red was used to stain the sebocytes of sebaceous gland and confocal microscopy was used for image acquisition. Primary antibodies used such as: CD34 (1:100, BD Pharmingen); α-6 integrin (1:100, BD Pharmingen); BrdU (1:250, Abcam); Ki67 (1:100; Novocastra); Filaggrin (1:1000, Abcam); Loricrin (1:1000, Abcam); K10 (1:1000, Abcam); Lrig1 (1:500, R&D systems) and Sox9 (1:500, Merck Millipore).

Antibodies included: β1 integrin (#610167), β4 integrin (#553745), α6 integrin (#555734) (all from BD Biosciences); Smad2 (#3103), phospho-Smad2 (S465/467, #3101), NF-κB p65 (#4764), phospho-NF-κB p65 (S536, #3033), E-cadherin (#4065), β-actin (#4967), Caspase-3 (#9662), Cleaved Caspase-3 (#9661), Cleaved Caspase-9 (#9509), cleaved PARP (#9544) (all from Cell Signaling), α-tubulin (#T5168, Sigma-Aldrich), ZO-1 (#sc-33725, Santa Cruz), and Alexa-Fluor®-conjugated secondary antibodies (Molecular Probes®, Life Technologies). Growth factors/hormones included: rhTGFβ1 (Invitrogen) and insulin (Sigma-Aldrich). The TβRI inhibitor SB-431542 was from InvivoGen.