Fluorescein isothiocyanateeconjugated anti mouse ccr3 antibody

The entire ocular surface, including the cornea and the conjunctiva up to the mucocutaneous junction, was dissected, as described previously. 31 Briefly, the entire eye was dissected together with the eyelid and fixed in paraformaldehyde for 1 hour. Next, under the dissecting microscope, the tissues were clipped to remove muscle and other accessory tissues so that only the conjunctival tissues remained. The conjunctival tissues were then blocked in 2% bovine serum albumin for 15 minutes, followed by permeabilization in 0.2% Triton X-100 for 15 minutes at room temperature. Subsequently, the conjunctival tissues were incubated with phycoerythrin (PE)-conjugated anti-mouse Siglec-F (552126; BD Biosciences, San Jose, CA), Alexa Fluor 488-conjugated anti-mouse CD125 antibody (558533; BD Biosciences), or fluorescein isothiocyanateeconjugated anti-mouse CCR3 antibody (144510; BioLegend, San Diego, CA) at 4 C overnight. The stained conjunctival tissues were washed three times with PBS and placed on glass slides to flatten them. A mounting medium containing 1 mmol/L DAPI (28718-90-3; Sigma-Aldrich, St. Louis, MO) was put on the conjunctival tissues. Images of the conjunctiva were analyzed using a DeltaVision microscopy imaging system (Applied Precision, Issaquah, WA).

Conjunctival tissues were cut into pieces and digested in 0.4% collagenase type I (C0130; Sigma-Aldrich) for 30 minutes. After washing with PBS, the digested suspension was passed through a 75-mm filter to obtain single cells. These single conjunctival cells were blocked in Flow Cytometry Staining Buffer (00-4222; eBioscience, San Diego, CA), which contains anti-CD16/32 antibody (1:100; 14-0161-85; eBioscience), at room temperature for 10 minutes and were then incubated in a mixture of the following antibodies (1:100) at room temperature for 30 minutes: allophycocyanin-conjugated anti-mouse CD45 antibody (559864; BD Biosciences), PE-conjugated anti-mouse Siglec-F antibody (552126; BD Biosciences), Brilliant Violet 421-conjugated anti-mouse CD64 antibody (139309; BioLegend), and either Alexa Fluor 488econjugated antimouse CD125 antibody (558533; BD Biosciences) or fluorescein isothiocyanateeconjugated anti-mouse CCR3 antibody (144510; BioLegend), or fluorescein isothiocyanateeconjugated anti-mouse CD34 antibody (553733; BD Biosciences). After washing with PBS, the stained cells were analyzed with a BD FACSCanto.