Anti mouse isotype

Lacrimal glands were surgically excised and immersed in 4% paraformaldehyde overnight at 4°C. The tissue blocks were washed, dehydrated, embedded in paraffin, cut to a thickness of 3 mm. The cells were counted that stained positively for CD4 (clone H129.9, 10μg/mL; BD Bioscience, San Diego, CA), CD8α (clone 53e6.7, 3.125μg/mL; BD Bioscience), CD11b (clone M1/70, 6.25 μg/mL; BD Bioscience),CD45 (clone 30-F11, 10 μg/mL; BD Bioscience), CD103 (clone 2E7, 10 μg/mL; Biolegend, San Diego, CA), paraffin sections were stained with the abovementioned primary antibodies and appropriate biotinylated secondary antibodies (all from BD Pharmingen, San Diego, CA) using a staining kit ( Vectastain Elite ABC kit; Vector, Burlingame, CA) and reagents (Nova-Red; Vector). Secondary antibody alone and appropriate anti-mouse isotype (BD Biosciences) controls were also performed. Two sections from each animal were examined and photographed with a microscope (Imager.Z1; Carl Zeiss Meditec, Oberkochen, Germany). Positively stained cells were counted in the stroma of the LG using image-analysis software (NIS Elements Software, version 3.0 BR; Nikon). Results were expressed as the number of positive cells per mm2.

Immunohistochemistry was performed to detect and count the number of cells in the conjunctival epithelium that stained positively for CD4 (clone H129.9, 2 μg/mL; BD Bioscience, San Jose, CA) and anti-rat IgG biotinylated secondary antibody (2 μg/mL goat anti-rat, 559286; BD Pharmingen, San Jose, CA)/Vectastain Elite ABC (using NovaRed reagents; Vector, Burlingame, CA), as previously described.^{21 (link),22 (link)} Secondary antibody alone and proper anti-mouse isotype (BD Biosciences) controls were also performed. Positively stained cells were counted in the goblet cell rich area of the conjunctiva using NIS Elements Software.