Cm163a

Routine immunofluorescence protocol was followed to stain VF basal cells with p63 (1:100; CM 163 A, Biocare Medical LLC, CA, USA). Unstained slides were deparaffinized in xylene and rehydrated in an ethanol series. Heat-induced antigen retrieval (HIER) was performed with 1X Citrate buffer (ph. 6.0; C999, Sigma, USA) at 121 °C for 15 min followed by 1 h of cooling at room temperature. The slides were blocked with a blocking buffer (1% Bovine serum albumin [BSA], 0.2% Triton-X-100, 10% Sodium Azide, 2% Donkey serum) for 1 h at room temperature. The primary antibody diluted in a solution containing 0.5% BSA, 0.2% Triton-X-100 and 10% Sodium Azide, was applied to the slides and left to incubate overnight at 4 °C in a humidified chamber. The slides were then incubated for 1 h after the application of donkey-anti-mouse secondary antibody conjugated to Alexa Fluor 647 (1:500; A-31571, Invitrogen, CA, USA). The slides were washed with 1X Tris-buffered saline-tween 20 (TBST) solution and treated with Hoechst nuclear counterstain (33258, Sigma, USA). The slides were then mounted with Dako aqueous mounting medium (10131720, Agilent Dako, CA, USA). The slides were viewed, and images were acquired at 20x by Axioimager microscope (Zeiss). p63 positive basal cells were counted in the right and left mid-membranous region of the VF across a fixed length of 300 μm using the counter tool in Fiji.