Anti aqp5

Double-label immunofluorescence was performed using amylase and AQP5 antibody. First, the paraffin-embedded submandibular gland sections were deparaffinised in different changes of xylene and rehydrated with different grades of ethanol. Antigens were retrieved using retrieval solution (DAKO) and treated with peroxidase and a protein block solution (DAKO) for 10 min each at RT. Sections were then incubated with primary antibodies anti-amylase (1:200, Santa Cruz Biotechnology, USA) or anti-AQP5 (1:200, Abcam, Cambridge, MA, USA) overnight at 4 °C. They were then washed in TBST two times and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (1:300, Sigma) or tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-rabbit secondary antibody (1:300), respectively, for 1 h at room temperature. This was followed by washing with TBST (5 min, 2 changes each) and mounted with Vectashield mounting medium (Vector Laboratories). Fluorescence was visualized by using FITC and TRITC channels in a confocal microscope (Olympus, Japan).

The formalin-fixed, paraffin-embedded submandibular gland tissue sections were sliced at 5 μm thickness, deparaffinised, and subjected to 1× Target Retrieval Solution, pH 6.0 (DAKO, Glostrup, Denmark). Sections were incubated with peroxidase blocking solution (DAKO) for 10 min at room temperature (RT). They were then washed with 1× TBST buffer (Scytek Lab, Logan, UT, USA) followed by a protein block (0.25% casein in PBS, DAKO) for 10 min at room temperature. Primary antibodies including anti-amylase (1:200, anti-mouse secondary antibody, Santa-Cruz Biotechnology), anti-AQP5 (1:100, anti-rabbit, Abcam, Cambridge, UK), anti-NHE1 (1:100, anti-rabbit, Santa-Cruz Biotechnology), anti-GRP78 (1:100, anti-mouse, Santa-Cruz Biotechnology), anti-GADD153/CHOP (1:100, anti-rabbit, Santa-Cruz Biotechnology, USA) or anti-PDI (1:100, anti-mouse, Enzo life sciences, Farmingdale, NY, USA) were diluted in antibody diluent provided by DAKO and incubated in a humidified chamber overnight at 4 °C. Slides containing tissue sections were further rinsed in TBST buffer and incubated with indicated secondary antibodies for 1 h at RT. AEC substrate chromogen (DAKO) was added and washed with deionized water. This was followed by counter staining with Mayer’s haematoxylin (Sigma-Aldrich). The slides were rinsed with tap water and mounted using an aqueous medium (Scytek Lab, USA).

Lung tissues were homogenized in RIPA buffer with the presence of protease inhibitors (Roche, Indianapolis, IN, U.S.A.), and protein concentrations were determined. Samples were loaded on to 10% gels for sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, U.S.A.) by means of the wet transfer method. The membranes were blocked with 5% nonfat milk, and then incubated overnight at 4°C with anti-AQP1 (Abcam, Massachusetts, U.S.A.), anti-AQP5 (Abcam, Massachusetts, U.S.A.), anti-BiP (Proteintech, Rosemont, U.S.A.) and anti-CHOP (Abcam, Massachusetts, U.S.A.) antibody. Anti-rabbit HRP–conjugated IgG was used to detect the bound antibodies. The binding of the specific antibody was visualized using enhanced chemiluminescence (Thermo Scientific, Tewksbury, MA, U.S.A.).