Rat anti mouse mhcii

Wild-type (mGlu4^+/+), and knockout mice lacking the Grm4 gene (mGlu4^−/−) in C57Bl/6 background were purchased from the Jackson Laboratories. (Strain 8815915) Animals were maintained in a UAB Animal Care Facility. All animal studies were performed following approved guidelines of the Institutional Animal Care and Use Committee. Breeding pairs were used to obtain neonates from both WT and mGlu₄ KO mouse colonies. Homozygous knockout status for the Grm4 gene was confirmed by genomic PCR. ADX88178 was synthesized at Vanderbilt as described in (Le Poul et al. 2012 (link); Yin et al. 2013 (link)). Primary antibodies used in the study were rat anti-mouse MHCII and iNOS from eBioscience. Biotinylated secondary antibody (anti-rat) and Alexa Fluor 488- Streptavidin were purchased from Jackson Immuno Laboratories. An enzyme-linked immunosorbent assay kit for mouse TNFα was purchased from R&D Systems (CA).

Conjunctival tissues excised from mice were fixed in 4% (v/v) paraformaldehyde (PFA, Sigma-Aldrich) for 1.5 hours and thoroughly washed. The samples were then blocked with 5% goat serum (Sigma-Aldrich) for 30 minutes and subsequently stained with rabbit anti-mouse ALDH1 (Abcam) and rat anti-mouse MHC-II (eBioscience) primary antibody in 5% (v/v) goat serum (Invitrogen) for 48 hours at 4°C in the dark. Samples were thoroughly washed and stained with goat anti-rabbit Alexa fluor 633 (Invitrogen) and goat anti-rat Alexa fluor 488 (Invitrogen) secondary antibody in 5% (v/v) goat serum and incubated for 1 hour at room temperature. Samples were thoroughly washed and mounted with DAPI-containing media (Vector Laboratories). Images were captured using a Leica SP5 (Leica Microsystems), followed by image analysis with Leica LAS AF lite software.