Icam 1

The lacrimal glands were cut into 6-µm sections using a microtome. The sections were fixed with pre-cooled acetone for 5 minutes, and then incubated with primary antibodies for TNF-α (Abcam Inc., Cambridge, MA), MMP-9 (Lifespan Biosciences Inc., Seattle, WA), ICAM-1, VCAM-1 (Bioss Inc., Woburn, MA), CD 4, IFN-γ (Novus Biologicals, Littleton, CO), IL-1β, IL-6, IL-17 (Abcam, Inc., Cambridge, UK), and IL-8 (Biorbyt, Cambridge, UK) for 1 hour at room temperature. After washing, the sections were incubated with the secondary antibody (DAKO Corp, Glostrup, Denmark) for 30 minutes. The immune reactions were visualized with diaminobenzidine chromogen, and the sections were counterstained with Mayer’s hematoxylin (Sigma) for 30 seconds at room temperature. The stained sections were photographed with a virtual microscope (NanoZoomer 2.0 RS, Hamamatsu, Japan). The calculated data were compared with the densitometry of the control group analyzed with ImageJ (National Institutes of Health, Bethesda, MD, USA).

The eyes and adnexa were surgically excised, embedded in optimal cutting temperature compound, and flash frozen in liquid nitrogen. Six micrometer sections were excised with a cryostat. The sections were fixed with pre-cooled acetone for 5 minutes, and the primary antibodies to TNF-α (Abcam Inc., Cambridge, MA), MMP-2 (Abcam Inc., Cambridge, MA), MMP-9 (Lifespan Biosciences Inc., Seattle, WA), ICAM-1 (Bioss Inc., Woburn, MA), and VCAM-1 (Bioss Inc., Woburn, MA) were added and incubated for 1 hour at room temperature. After washing, the sections were incubated with secondary antibody (DAKO Corp, Glostrup, Denmark) for 30 minutes. Immunoreactions were visualized with diaminobenzidine chromogen, and the sections were counterstained with Mayer’s hematoxylin (Sigma-Aldrich) for 30 seconds at room temperature. Images of the sections were photographed with a Virtual Microscope (NanoZoomer 2.0 RS). The stained cells were evaluated oven a range of 0.1 mm² in the lacrimal gland of each group.

Total protein was extracted from cells with a Cell Protein Extraction Kit (Shanghai Epizyme Biomedical Technology, China), and the protein concentration was determined with the bicinchoninic acid protein detection kit (Beyotime Biotechnology, China). Total protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. Antibodies against the following targets were added and incubated overnight at 4°C: CX3CL1 (Abcam, UK; ab85034, 1:900), VCAM-1 (bs-0396R, 1:1000), ICAM-1 (bs-6326R, 1:1000), GAPDH (bs-2188R, 1:5000), p65 (bs-23217R, 1:1000), p-p65 (bs-0982R, 1:1000), IκBα (bs-1287R, 1:1000), p-IκBα (bs-18128R, 1:1000) and lamin-B (bs-1840R, 1:1000) (all Bioss, Beijing, China). After incubating with horseradish peroxidase-conjugated AffiniPure goat anti-rabbit immunoglobulin G antibody, a chemiluminescence reaction was performed, and the intensity was analyzed with ImageJ software. Each western blot result shown is representative of three independent experiments. Please refer to the Blot Transparency section of supplementary information (Fig. S6) for images of all uncropped western blots in this study.