Each conjunctival biopsy was obtained from the inferotemporal bulbar conjunctiva quadrant in an area of clinically apparent conjunctival inflammation. Specimens from control eyes were obtained at the time of surgery from the same conjunctival location. The average size of specimens was 10 × 5 mm. Tissue was stored at −80°C, embedded in OCT medium, before slide preparation and immunostaining. Conjunctival biopsies recovered from stock were sliced into 5 μm sections and placed on poly-L-lysine–coated slides for immunostaining on frozen sections. All slides were fixed in precooled acetone for 10 minutes. Nonspecific binding was blocked by 30 minutes of incubation with 5% goat serum at 37°C, except for IL-13, which was blocked with 5% rat serum. Sections were incubated overnight at −4°C with primary antibodies (goat anti-human IL-1, −6, −12, −17; RD Systems, Inc., Boston, MA; and rat anti-human IL-13; Abcam, Cambridge, MA;
Table 1). After 3 washes with PBS with BSA 0.1 M, all specimens were blocked with 5% rabbit serum for 30 minutes. Next, all slides were incubated with biotinylated-secondary antibodies (Ab) directed to IL-1, IL-6, IL-12, IL-13, and IL-17 for 1 hour (
Table 1), washed with PBS-BSA, and incubated for 30 minutes with streptavidin peroxidase (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). The reaction products were developed with a mixture of 3,3-diaminobenzine-4 HCL (DAB). The last step was staining with Histotik for 6 seconds, stopping the reaction with distilled water. Collagen IV was used as a positive control for primary Ab, while PBS only and secondary Ab without primary Ab were used as negative controls for the internal process. Each section was mounted and examined at 30× magnification using light microscopy (model Olympus BX 41; Olympus, Center Valley, PA). For each sample, two independent observers counted stained cells and the results were scored using a semiquantitative scale 0 to 3, with 0+ indicating less than 5 cells per field, 1+ between 5 and 25 cells per field, 2+ between 25 and 50 cells per field, and 3+ more than 50 cells per field.
Conjunctival specimens also were stained with hematoxylin and eosin, periodic acid-Schiff and Giemsa, and analyzed by light microscopy. Histologic characteristics were noted for the presence or absence of inflammatory cells and microangiopathy. Microangiopathy was defined as ultrastructural changes, such as reduction of the vascular space, vascular basement membrane thickening, change in conjunctival vessel architecture, and perivascular inflammatory infiltrate.