Alexa fluor 546 and 488 conjugated goat iggs

Freshly isolated keratinocyte subpopulations were fixed in 4% buffered PFA and cytospun onto glass slides. Cells were permeabilized with Triton × 100 0.1%, stained for K10 (clone EP1607Y, Epitomics, Burlingame, CA, USA), K15 (EPR1614Y, Epitomics), and Involucrin (clone I9018, Sigma) and then labeled with Alexa Fluor secondary antibodies, Alexa Fluor 546 and 488-conjugated goat IgGs (Invitrogen, Waltham, MA, USA). Then slides were stained with 1 μg/mL Dapi (Sigma). Immunofluorescence on skin equivalent slides was performed as in IHC methods except for the different primary antibodies: K10, K15, and Survivin (Novus Biologicals, Littleton, CO, USA) and the secondary antibodies Alexa Fluor 546 and 488-conjugated goat IgGs (Invitrogen). Micrographs were taken on a Confocal Scanning Laser Microscopy (Leica TCS4D) (Leica, Exton, PA, USA). Quantification of immunofluorescence staining was performed by analyzing 6 representative fields for each staining sample, using ImageJ software. Scoring was made by means of positive cell counting.