Ab18586

Tissue sections were immersed in Citrate Buffer solution (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) and placed in a water bath at 90°C for 15 minutes to break protein cross‐linking to enhance staining intensity. Tissues were then rinsed with 0.1% PBS Tween 20 and permeabilised in 0.2% Triton X‐100. Sections were then blocked with 0.25% Gelatin from cold water fish and 10% foetal bovine serum for 60 minutes. Primary antibodies were prepared in PBS: anti‐Ki67 (1:400; rabbit polyclonal, ab15580, abcam), anti‐cytokeratin 6 (1:100; mouse monoclonal, ab18586, abcam), and anti‐cytokeratin 14 (1:100; mouse monoclonal, ab7800, abcam). Sections were incubated with the primary antibody overnight at 4°C followed by secondary antibody (Alexa Fluor 488 goat anti‐rabbit IgG [1:400; 10729174, Fisher Scientific] or DyLight 488 goat anti‐mouse IgG [1:400; ab96879, abcam]) at room temperature for 1 hours. Nuclei were stained with Hoechst (1:10 000) and mounted using Citifluor (Glycerol/PBS solution, Citifluor Ltd) and sealed with nail varnish.

Human eyelid sections and HMGECs were fixed with cold methanol for 15 minutes at −20°C. Following three phosphate‐buffered saline (PBS) rinses for 5 minutes each, samples were blocked with 2% bovine serum albumin (BSA, Sigma‐Aldrich Corp., St. Louis, MO) in PBS for 60 minutes, and then incubated overnight at 4°C in a moist chamber with antibodies specific for Lrig1 (ab214102,1:100), cytokeratin 14 (ab181595, 1:500), cytokeratin 6 (ab18586, 1:500), DNase2 (ab8119, 1:100; Abcam, Cambridge, MA), or lysosomal‐associated membrane protein 1 (LAMP‐1;H4A3, 1:15; Developmental Studies Hybridoma Bank, Iowa City, IA), or the BSA diluent. After three additional PBS rinses, donkey anti‐rabbit (ab150075, 1:500, Abcam) or donkey anti‐mouse (2492098, 1:500, EMD Millipore, Temecula, CA) secondary antibodies were applied for 1 hour at room temperature. For neutral lipid staining, some eyelid sections were fixed in 4% paraformaldehyde for 15 minutes. Following additional washes, samples were exposed to LipidTOX Green neutral lipid stain (1:500, Thermo Fisher Scientific) in a humid chamber for 30 minutes. Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, 1 μg/mL, Sigma‐Aldrich) and samples were covered with VectaMount mounting medium (Vector Laboratories, Burlingame, CA), and observed with a confocal microscope (Leica TCS SP8, Leica Microsystems, Wetzlar, Germany).