Dmi6000b brightfield microscope

Extraorbital lacrimal glands from six-, 10- and 16-week-old NOD.H-2^h4 and NOD.H-2^h4 DKO mice of both sexes were harvested for 5 µm paraffin sectioning and stained with hematoxylin and eosin (H&E). Focus scores were determined from stitched images of lacrimal glands by quantifying lymphocytic foci (>50 mononuclear cells) per 4 mm² tissue. Images were obtained with a Zeiss Axiovert 200M brightfield microscope with a ×10 objective, and the total lacrimal gland area was determined using MetaMorph software. Sections from 16-week-old mice were stained with anti-CD45 or anti-MMP-9 antibodies. Images were obtained using a Leica DMI6000B brightfield microscope and LASX software.

After cervical dislocation, intact eyes with eyelids were harvested from mice and fixed overnight at 4°C in 4% (v/v) paraformaldehyde in phosphate-buffered saline solution (PBS), washed three times in PBS and placed in 70% (v/v) ethanol for 24 hours. Samples were embedded in paraffin and 5 µm sections were stained with periodic acid-Schiff (PAS) reagent. Goblet cell density continuous with superior and inferior palpebral conjunctiva and conjunctival fornices in two 100 µm PAS-stained sections was determined using a Zeiss Axiovert 200M brightfield microscope with a 10X objective. Sections from 16-week-old mice were stained with anti-CD45 (Biolegend, San Diego, CA) or anti-MMP-9 (Abcam, Cambridge, UK) antibodies. All images were obtained using a Leica DMI6000B brightfield microscope and LASX software.