Rabbit anti loricrin

The following antibodies were used in this study: mouse anti-p63 (Santa Cruz), rabbit anti-cytokeratin 5 (Abcam), rabbit anti-integrin β1 (Abcam), rabbit anti-loricrin (Covance), goat anti-involucrin (Santa Cruz), mouse anti-tubulin α (Developmental Studies Hybridoma Bank), mouse anti-BrdU (Roche), rabbit anti-Smad2/3 (Cell Signaling), Alexa 488-goat anti-mouse IgG, Alexa 488-goat anti-rabbit IgG, Alexa 594-goat anti-rabbit IgG, R-phycoerythrin-goat anti-mouse IgG (Molecular Probes), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (KPL), HRP-conjugated rabbit anti-goat IgG (Invitrogen), and HRP-conjugated goat anti-rabbit IgG (Cell Signaling).

For immunofluorescent detection of markers of the epidermis, sections were deparaffinized and rehydrated in reducing concentrations of ethanol. Sections were subjected to antigen retrieval by boiling in 10mM Sodium citrate (pH6.0) for 30 minutes. Tissues sections were permeabilized using 0.5% Triton X-100 followed by blocking in blocking solution (10% normal goat serum, 0.1% Bovine Serum Albumin in 1X Phosphate Buffered Saline (PBS)) for one hour at room temperature. Sections were then incubated in anti-mouse F′ (ab) fragment (Jackson ImmunoResearch Laboratories, West Grove, PA) for five minutes to reduce non-specific binding of anti-mouse secondary. Sections were incubated with 1:100 rabbit anti-Irf6 (Sigma-Aldrich; St. Louis, MO), 1:250 rabbit anti-Keratin 14 (Covance), 1:150 mouse anti-p63 (Santa Cruz), and 1:250 rabbit anti-loricrin (Covance) overnight at 4°C, followed by incubation with either goat anti-mouse AlexaFluor 488 or goat anti-rabbit AlexaFluor 555 (Life Technologies, Grand Island, NY). Nuclei were counterstained for 10 minutes in a 1:10,000 dilution of 4′ 6-diaminidino-2-phenylindole (DAPI; Life Technologies, Grand Island, NY). Slides were then mounted in ProLong GOLD Antifade reagent (Life Technologies, Grand Island, NY). Images were taken using a Nikon Eclipse 90i fluorescent microscope.