Skin was embedded in OCT (optimal cutting temperature) (Tissue-Tek). Samples were sectioned at 4- to 6-μm sections using CM3050S cryostat (Leica Microsystems GmbH). For the staining on frozen sections, tissues were fixed in 4% paraformaldehyde for 10 min at room temperature, and then washed in phosphate-buffered saline (PBS). Nonspecific antibody binding was blocked with 5% horse serum, 1% bovine serum albumin, and 0.2% Triton X-100 during 1 hour at room temperature. Primary antibodies were incubated overnight at 4°C in blocking buffer. Sections were rinsed in PBS and incubated with secondary antibodies during 1 hour at room temperature. Nuclei were stained with Hoechst (4 mM). Slides were mounted using Glycergel (Dako) supplemented with 2.5% DABCO (Sigma-Aldrich). For the staining on paraffin sections, 4-μm paraffin sections were deparaffinized and rehydrated. Antigen unmasking was performed in citrate buffer (pH 6) at 98°C for 20 min using the PT (pre-treatment) module. Endogenous peroxidase was blocked using 3% H2O2 (Merck) in methanol for 20 min at room temperature. Endogenous avidin and biotin were blocked using the Endogenous Blocking kit (Invitrogen) for 20 min at room temperature. Primary antibodies were incubated overnight at 4°C. Anti-mouse biotinylated secondary antibodies, as well as Standard ABC kit, and ImmPACT DAB (Vector Laboratories) were used for the detection of HRP activity. Slides were mounted using SafeMount (Labonord).

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