Rabbit anti keratin 10

Section slides were fixed with 4% paraformaldehyde (Merck, Martillac, France), permeabilized with Tris-buffered saline (PBS) (Fisher Scientific, Illkirch-Graffenstaden, France) in the presence of 0.3% triton (Euromedex, Souffelweyersheim, France) and blocked for 1 h with PBS 5% BSA (Euromedex, France) plus 10% goat serum (Millipore, France). Sections were then incubated overnight at 4 °C with mouse anti-E-cadherin antibody (1/200, Becton Dickinson), rabbit anti-Keratin 1 (1/200, Abcam), rabbit anti-Keratin 10 (1/200, Abcam), rabbit anti-Keratin 5 (1/200, Abcam), and rabbit anti-Keratin 14 (1/200, Novus). After three PBS washes, the skin was incubated with secondary antibodies, Alexa 488 goat anti-mouse IgG (H + L) and Alexa 594 goat anti-rabbit IgG (H + L), respectively. After subsequent washing, the sections were mounted with Prolong Gold antifade reagent containing DAPI (Thermofisher Scientific, Illkirch-Graffenstaden, France), and images were acquired using Nikon confocal A1R (Nikon, Tokyo, Japan).

For immunofluorescence staining, tissues were embedded in Tissue-Tek O.C.T Compound (Sakura Finetek, CA, USA) and 6 µm thick sections were fixed in acetone at −20 °C before staining. The following antibodies were used and incubated in a dark room at room temperature for 45 min: rabbit anti-involucrin (Abcam, Cambridge, MA, USA), rabbit anti-filaggrin (Abcam, Cambridge, MA, USA), rabbit anti-keratin 10 (Abcam, Cambridge, MA, USA), mouse anti-Ki67 IgG1 (BD Biosciences, CA, USA), rabbit anti-AC9 (ab191423, Abcam, Cambridge, MA, USA) and rabbit anti-beta 2 adrenergic receptor (ab61778, Abcam, Cambridge, MA, USA). Tissues were then incubated with Alexa Fluor 488 donkey anti-rabbit IgG or Alexa Fluor 594 goat anti-mouse IgG (1:1600, Thermofisher Scientific, CA, USA) for 30 min also at room temperature. Nuclear counter staining using DAPI (SouthernBiotech, AL, USA) was then effected on different samples. Each tissue was observed using a Zeiss Axio Imager M2 microscope with an AxioCam ICc1 camera. The quantification of immunofluorescence staining was performed by densitometry using ImageJ software (from Wayne Rasband, National Institute of Health (NIH), USA).