Immunohistochemical Analysis of Salivary Gland

Immunohistochemistry was performed in formalin-fixed, paraffin-embedded submandibular gland tissue as described previously [21 (link)]. Antibodies analyzed with immunohistochemistry were alpha-amylase (1:200, anti-mouse, Santa-Cruz Biotechnology, Dallas, TX, USA), aquaporin-5 (1:150, anti-rabbit, Abcam, Cambridge, UK), 4-Hydroxy-2-nonenal (4-HNE) (1:100, anti-rabbit, Abcam, Cambridge, UK), GRP78 (1:100, anti-mouse, Santa-Cruz Biotechnology, Dallas, TX, USA), and GADD153/CHOP (1:100, anti-rabbit, Santa-Cruz Biotechnology, Dallas, TX, USA). Double-label immunofluorescence was performed using anti-goat IP3R2 (1:100, Santa-Cruz Biotechnology, Dallas, TX, USA) and anti-rabbit AQP5 antibody (1:300, Abcam, Cambridge, UK). Sections were incubated with primary antibodies in a humidified chamber overnight at 4 °C. Slides were then rinsed in 1×TBST buffer (DAKO) three times and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-goat secondary antibody (1:300, Sigma) or TRITC-conjugated anti-rabbit secondary antibody (1:300), respectively, for 1 h at room temperature. Slides were then followed by washing with TBST (3 times) and mounted with DAPI (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence was visualized using FITC and TRITC channels in a laser scanning confocal microscope (Olympus, Japan).