Skin tissue samples or RHE were fixed in 4% formaldehyde, embedded in paraffin, and 5‐μm sections were cut. Antigen retrieval was performed in 10 mmol/L citrate buffer pH 6.0 for 30 min at 95°C. The activity of endogenous peroxidase was quenched with Bloxall blocking solution (Vector Laboratories). Samples were stained using the R.T.U. Vectastain Kit with the ImmPACT AMEC Red Substrate Kit (Vector Laboratories) according to the manufacturer's protocol. Sections were counterstained with Mayer's haematoxylin solution, mounted with Glycerol Mounting Medium (Dako). Double staining was made using the ImmPRESS Duet Double Staining Polymer Kit, according to the manufacturer's protocol (Vector laboratories).
For immunofluorescence, sections were blocked with 3% BSA and 5% normal goat serum in PBS for 1 h. Next, sections were incubated for 2 h with primary antibodies followed by 1 h incubation with Alexa Fluor 488‐conjugated donkey anti‐rabbit and Alexa Fluor 555‐conjugated donkey anti‐mouse IgG secondary antibodies (Invitrogen) and mounted with DAPI Fluoromount‐G (SouthernBiotech).
For immunofluorescence, sections were blocked with 3% BSA and 5% normal goat serum in PBS for 1 h. Next, sections were incubated for 2 h with primary antibodies followed by 1 h incubation with Alexa Fluor 488‐conjugated donkey anti‐rabbit and Alexa Fluor 555‐conjugated donkey anti‐mouse IgG secondary antibodies (Invitrogen) and mounted with DAPI Fluoromount‐G (SouthernBiotech).