Rabbit anti filaggrin

Immunofluorescence in tissues was performed as described previously^{16 (link),37 (link)–39 (link)}. Briefly, the skin tissues were embedded, fixed, permeabilized, incubated with primary mouse anti-K5 (1:200; Progen Biotechnik, Heidelberg, Germany), rabbit anti-Filaggrin (1:1000; Abcam, Cambridge, MA), rabbit anti-E-cadherin (1:500; Cell Signaling, Danvers, MA), mouse anti-β-catenin (Cell Signaling, Danvers, MA), rabbit anti-F-actin (Cell Signaling, Danvers, MA) and rabbit anti-ZO-1(Cell Signaling, Danvers, MA), and then incubated with Alexa Fluor 488 F (ab’) 2 fragments of goat anti-guinea pig IgG antibodies and Alexa Fluor 568 of goat anti-rabbit IgG antibodies (Invitrogen, Carlsbad, CA). The cells were then fixed in Prolong Gold Antifade with DAPI (Invitrogen, Carlsbad, CA) to visualize the cell nuclei, and observed under a fluorescence microscope (OlympusIX71, Japan) with a peak excitation wavelength of 340 nm.

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).