Ten μL drops of 1 mg/mL goat anti-rabbit IgA conjugated to fluorescein (Bethyl Laboratories, Montgomery, TX) were added under the superior and inferior conjunctiva six times at 10-minute intervals. The tissue was then isolated, fixed for 2 hours in 2% paraformaldehyde in HWB (70 mM NaCl, 30 mM HEPES, 2 mM CaCl
2, pH 7.4) + 300 nM 4′,6-diamidino-2-phenylindole (DAPI). The inclusion of fluorescent nuclear dyes such as DAPI simplifies identification of follicles using fluorescent stereomicroscopy.
9 Isolated follicles, along with unexposed control follicles, were dehydrated with a series of increasing concentrations of ethanol, infiltrated in butyl-methylmethacrylate (BMMA) resin, and polymerized at 4°C. For post-embedding immunocytochemistry, a goat anti-rabbit IgA (Bethyl Laboratories) was biotinylated using an NHS-PEO
4-biotinylation kit according to the manufacturer's instructions (Pierce, Rockford, IL). Sections of 0.5 μm were etched for 10 minutes in acetone before antigen retrieval by exposure to 100 mM glycine (pH 9.6) at 98°C for 30 minutes, blocked with 1% bovine serum albumin (BSA) in HWB for 1 hour, and stained overnight with 20 μg/mL biotinylated goat anti-rabbit IgA, followed by 4 hours in 5 μg/mL streptavidin-Alexa 488 (Invitrogen, Carlsbad, CA) + 300 nM DAPI.