Anti amylase

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).

Immunoblotting was performed using submandibular gland homogenates as described previously [21 (link)] to determine the expression of alpha-amylase, PDI, GRP78, ATF-4, CHOP, sXBP-1, p-JNK, and JNK. The antibodies used for western blotting analysis were anti-amylase (1:1000, anti-mouse, Santa-Cruz Biotechnology, Dallas, TX, USA), anti-PDI (1:1000, clone 1D3, Enzo Life Sciences, Farmingdale, NY, USA), anti-GRP78 (1:2000, anti-mouse, Santa-Cruz Biotechnology, Dallas, TX, USA), anti-CHOP (1:2000, anti-mouse, Cell Signaling, MA, USA), anti-XBP1 (1:1000, anti-mouse, Santa-Cruz Biotechnology, Dallas, TX, USA), anti-p-JNK (1:1000, anti-rabbit, Cell Signaling, MA, USA), JNK (1:1000, anti-rabbit, Cell Signaling, MA, USA), anti-ATF-4 (1:1000, anti-rabbit, Cell Signaling, MA, USA), and anti-actin (1:5000, anti-rabbit, Cell Signaling, MA, USA).

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).