Tissues were embedded in optimal cutting temperature freezing compound (OCT, Tissue-Tek; Sakura Fintek, Torrance, CA), and 4-μm-thick sections were cut. The tissue was fixed with −20°C acetone for 10 minutes, followed by incubation for 1 hour with monoclonal antibodies to keratin-4 (Sigma-Aldrich), -19 (DakoCytomation, Carpinteria, CA), and -3 (AE-5 antibody was a kind gift from Tung-Tien Sun, New York University, New York, NY). Normal mouse immunoglobulin (Sigma-Aldrich) and pancytokeratin (AE-1 and AE-3; Sigma-Aldrich) were used as the negative and positive controls, respectively. The cells were subsequently incubated with secondary antibody (1:200 biotinylated horse anti-mouse immunoglobulin G) for 1 hour, detected with the mouse immunoperoxidase detection kit (Vectastain Elite Kit; Vector Laboratories, Burlingame, CA), and stained with 3,3′-diaminobenzidine (DAB) substrate (Sigma-Aldrich).
The expression of conjunctival goblet cell mucin, MUC5AC, was determined by RT-PCR.
26 27 28 Total RNA was isolated from cultured conjunctival epithelial cells by using a guanidinium isothiocyanate protocol (RNeasy; Qiagen, Valencia, CA) and subjected to RNase-free DNase I digestion, extracted twice with phenol-chloroform-isoamyl alcohol (24:24:1), precipitated with ethanol, dissolved in RNase-free water, and quantified spectrophotometrically. One microgram of total RNA was used for cDNA synthesis (SuperScript II reverse transcriptase; Invitrogen, Carlsbad, CA). PCR was performed with primers for human MUC5AC.
26 The primer sequences were as follows: MUC5AC sense primer, 5′-TCCACCATATACCGCCACAGA-3′, and antisense primer, 5′-TGGACCGACAGTCACTGTCAAC-3′. The amplification reaction was performed in a thermal cycler (PCR Sprint; Thermo Hybaid, Ashton, UK). The conditions were: 3 minutes at 96°C, followed by 30 cycles of denaturation for 45 seconds at 96°C, amplification for 1 minute at 55°C, and extension for 1 minute at 72°C. The predicted length of the PCR product was 103 bp.
27 Amplified cDNA was analyzed by electrophoresis on a 1% agarose gel and viewed by ethidium bromide staining. Total RNA from forniceal conjunctival epithelium was used as a positive control. Actin PCR was conducted at the same time as a system control.