Epitope retrieval buffer

Skin tissue sections were deparaffinized in xylene and dehydrated in graded alcohol. After washing the samples in PBS, sections were placed in epitope-retrieval buffer (DakoCytomation, Carpenteria, CA, USA) at 95°C for 20 min and subsequently cooled to room temperature (RT) for an additional 20 min. The sections were blocked with 10% goat serum in PBS, followed by blocking for endogenous peroxidases stained with peroxidase block solution (DakoCytomation). Sections were incubated overnight at 4°C with antibodies against anti-CD3 and anti-Orai-1 (Cell Signaling Co., Danvers, USA). Unbound antibodies were removed the following day by washing the slides three times with PBS. Areas positive for CD3 and Orai-1 induction were stained brown after development with diaminobenzidine. The slides were counterstained with filtered Mayer's hematoxylin (Sigma-Aldrich, St. Louis, MO), rinsed with distilled water, allowed to dry, and then mounted for viewing purposes. The images were observed using a Leica digital camera and microscope (Leica Co., Wetzlar, Germany).

Samples were soaked in xylene twice for 5 min to remove paraffin, and then 100% ethanol twice 3 min to hydrophilize. After washing with water in several minutes, the samples were soaked into epitope retrieval buffer (DAKO) and autoclaved at 105 °C for 10 min. The samples were then soaked into 1× PBS for several minutes and incubated with 100 μL of primary antibody in IMMUNO SHOT (Cosmo Bio) at 4 °C overnight. Antibodies and dilutions are shown in Supplementary Table 2. After two 5-min washes with PBS, the samples were incubated for 30 min at room temperature with secondary antibodies. The samples were washed twice in PBS for 5 min, and then nuclear staining was performed with hematoxylin for 10–30 s. After washing with water in 10 min, samples were placed in 100% ethanol (three times), and then placed in xylene three times. Finally, the samples were evaluated under a microscope (Keyence).