Anti gadd153 chop

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).

Immunocytochemistry was performed as described previously with some modifications^{18 (link)}. Cells were fixed with 4% paraformaldehyde/PBS and permeabilized with 0.5% Triton X-100/PBS for 15 min. Then, cells were blocked with 5% foetal bovine serum/PBS for 30 min. For primary antibodies, cells were incubated at 4 °C for 16 h with the respective antibodies, such as anti-SOX1 (1:100, R&D Systems), anti-SOX2 (1:100, R&D Systems), anti-PAX6 (1:100, Stemgent, San Diego, CA, USA), anti-Nestin (1:200, eBioscience, San Diego, CA, USA), anti-TUJ1 (1:1,500, Covance, Berkeley, CA, USA), anti-MAP2 (1:1,500, Millipore), anti-TBR1 (1:200, Abcam, Cambridge, UK), anti-GFAP (1:1,500, DakoCytomation, Carpinteria, CA, USA), anti-p62 (1:100, Abcam), anti-NeuN (1:100, Millipore), anti-cleaved caspase 3 (1:800, Cell Signaling Technology, Beverly, MA, USA), anti-GRP78/BIP (Abcam), or anti-GADD153/CHOP (Santa Cruz Biotechnology). Cells were washed with PBS and then incubated for 60 min with secondary antibodies such as Alexa Fluor 488, 555, 594, or 647 (Thermo Fisher Scientific). Nuclei were counterstained with Hoechst 33342 (Dojindo, Kumamoto, Japan). The cytoplasm was stained with HCS CellMask Deep Red Stain (Thermo Fisher Scientific).