Mhc class 2

To prepare frozen sections of conjunctiva, eyes were enucleated with the lids intact, fixed in 4% formaldehyde in phosphate buffered saline (PBS) for 48 h, followed by cryoprotection in 10%–30% sucrose for 72 h before embedding in OCT. Tissue sections 7 µm in thickness were cut using a microtome and placed on slides to be stored at – 20°C until ready to use. Conjunctival explants were fixed in 4% formaldehyde before staining. Tissue sections and explants were blocked by PBS that contained normal goat, hamster, or donkey serum, 0.3%–1% Triton X-100 at RT for 1 h, and incubated overnight at RT with primary specific antibodies for CD11c (Biolegend, Dedham, MA), MUC5AC (Abcam, Cambridge, MA), MHC class II (BD Biosciences, San Jose, CA), or TSP-1 (Santa Cruz Biotech, Santa Cruz, CA). After a PBS rinse, fluorescence-conjugated secondary antibodies (Invitrogen, Thermo Fisher Scientific, Waltham, MA) were applied for 1 h in RT. Cell nuclei were counterstained with DAPI dye. Fluorescent staining in tissue sections was visualized using the FSX100 Olympus fluorescence microscope. Staining in conjunctival explants was visualized using a Zeiss LSM 700 confocal microscope equipped with a 63× oil objective. Images (z-stacks) were captured using the Zeiss ZEN software. Microscopic images were analyzed using NIH ImageJ software (14 (link)).

Mouse IgG2a anti-Ft. LPS mAb used to generate mAb-iFt immune complexes was purchased from Fitzgerald (Cat# 10-F02, clone#M023621, Acton, MA). The following flow cytometry antibodies were purchased from BD Biosciences (San Jose, California): F4/80 (PE), CD11b (FITC), CCR7 (PE-Cy5.5), MHC class II (APC), B7.1 and B7.2 (PercP), CD11c (APC)

Monoclonal antibodies (mAbs) against CD4, CD11b, CD25, CD40, CD80, CD86, F4/80, Gr-1, and I-Ab (MHC class II) were purchased from BD PharMingen (San Diego, CA, USA), and mAbs against B7-H1 and Foxp3 were purchased from eBioscience (San Diego, CA, USA). Intracellular staining protocols for regulatory T cells were followed for Foxp3 staining. For carboxyfluorescein succinimidyl ester (CFSE) labeling, splenic T cells (10⁷/ml) from BALB/c mice were incubated with 0.5 μM of CFSE (Invitrogen, San Diego, CA, USA) for 10 min at room temperature. Flow analyses were performed with a BD FACSCanto II flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA).

Cultured conjunctival goblet cells were stained with eFluor 780-conjugated Fixable Viability Dye (eBioscience, San Diego, CA). TGF-ß1 (R&D Systems), TGF-ß2 (R&D Systems), TSP-1 (Santa Cruz Biotech.), CD36 (eBioscience), and MHC class II (BD Bioscience, San Jose, CA) primary antibodies and AlexaFluor-conjugated secondary antibodies (Life Technologies) were used to assess surface staining. Their respective isotype-matched antibodies served as negative controls. Intracellular TGF-ß1, TGF-ß2 and TSP-1 were stained with the same antibodies but using an intracellular staining kit (eBioscience) as per the manufacturer’s instruction. Fluorescence-labeled cells were analyzed using BD LSRII Flow Cytometer (BD Bioscience). Further analysis of the data was performed using FlowJo v9.4.10 software (Tree Star, Inc., Ashland, OR).

All steps were done on ice or in 4°C centrifuges to prevent the sudden upregulation of MHC class II caused by handling [25 (link)]. Plates containing DC cultures were cooled for 20 min. The cells were then gently harvested, spun, counted and distributed into 96 well plates at about ~10⁶ per well. We added 10 μl “ultrablock” (a mixture of 1:1:1 mouse, rat and goat serum containing 10μg/ml of the monoclonal anti-FcR antibody 2.4G2) for 10 min before adding a master-mix of a combination of antibodies anti-mouse CD80, CD40, CD11c and MHC class II (BD Pharmingen). Each antibody was titrated for optimal signal-to-background before use. After 20 min, the cells were washed and resuspended in PBS containing the viability dye, 7AAD plus 1% FCS, for 5 min, then centrifuged and resuspended in FACS buffer for analysis.