Corneas dissected from postmortem human fetal and adult donors were either fixed in 10% neutral formalin and paraffin embedded or frozen in liquid nitrogen-cooled 2-methylbutane (OCT tissue-embedding medium; Tissue-Tek; Sakura Finetek, Tokyo, Japan) and stored at −80°C. Frozen sections (8 μm) cut with a cryostat (Microm HM 500; Carl Zeiss Meditec, Inc., Dublin, CA) were collected on electrostatically charged slides (Superfrost; Fisher Scientific, Pittsburgh, PA). Cultured limbal explant epithelial cells (serial passages: Pa1, Pa2, Pa3, Pa4, and Pa5) were centrifuged and revived overnight in serum-free media for 6 hours and the cell pellets were fixed in 10% neutral formalin overnight and paraffin embedded. Paraffin-embedded tissue (4 μm) sections on silane-coated slides were dewaxed in xylene (BDH, Poole, UK) and rehydrated in changes of 100% ethanol ′ 2 and 70% ethanol ′1, and double distilled water (ddH2O; MilliQ, Millipore Ltd., Tokyo, Japan). Cryostat sections were fixed in ice-cold 100% methanol for 10 minutes, air dried, and stored at −80°C. Before immunostaining, the sections were thawed and preincubated in 0.1% Triton X-100, 1× PBS for 1 hour at room temperature.
Paraffin-embedded fetal and adult human corneal tissue and limbal epithelial explant cell pellets of Pa1 to Pa5 samples on silane-coated slides were dewaxed and rehydrated as just described. A heat-mediated (750-W microwave) antigen retrieval method using a sodium citrate (0.1 M, pH 6.0, three times for 5 minutes each) bath was used. Slides cooled at room temperature for 5 minutes were then washed twice with TBS (Tris-buffered saline). Endogenous peroxidase was blocked with 3% H2O2 (Ajax Finechem, Australia) in methanol (APS, Australia) for 10 minutes. After the slides were washed twice for 5 minutes with 1× TBS, 20% normal goat serum (Vector Laboratories, Burlingame, CA) in 2% BSA in 1× TBS (bovine serum albumin) was used to prevent nonspecific staining by incubating the slides for 30 minutes at room temperature. Slides were then incubated with the primary antibody: LHK 15 clone primary mouse IgG2a anti-human CK15 (Biocare Medical, Concord, CA) in the final concentration of 2 μg/mL, ON359 clone primary mouse IgG3 anti-human CK14 (U.S. Biological, Swampscott, MA) in a final concentration of 1 μg/mL, and 3G51 clone primary mouse IgG1 anti-human P-Cadherin (U.S. Biological) in a final concentration of 2 μg/mL were used as the primary antibodies (used on formalin-fixed paraffin-embedded sections). Sections were incubated with the primary antibody for preoptimized times (60 minutes at 37°C) and then washed three times for 5 minutes each with 1× TBS and then incubated with an appropriate secondary antibody (goat anti-mouse; Dako) for 30 minutes at room temperature. After three slide washes with 1× TBS (5 minutes), the slides were incubated with 1:100 diluted streptavidin conjugated to horseradish peroxidase (Vector Laboratories, Inc.) for 60 minutes at room temperature. After three washes with 1× TBS for 5 minutes, AEC (3-amino-9-ethylcarbazole; Sigma-Aldrich) was used as a substrate chromogen. One microliter of 30% H2O2 used to initiate the reaction was added to the AEC and then placed on the slides for 5 minutes; a positive reaction indicated by a dark red color. The slides were counterstained with Meyer hematoxylin (Dako) for 20 seconds.
Cryostat corneal sections on silane coated slides were thawed and preincubated in 0.1% Triton X-100 in 1× TBS for 60 minutes at room temperature and then fixed for 10 minutes in acetone. Endogenous peroxidases were blocked with 3% H2O2 (Ajax Finechem, Seven Hills, NSW, Australia) in methanol (APS) for 10 minutes. The slides were washed for 5 minutes twice with 1× TBS. Incubation with 20% normal rabbit serum (Vector Laboratories, Inc.) for 30 minutes at room temperature was used to prevent nonspecific staining before incubation with Wnt-4 primary antibody. Tissue slides were incubated with 1:50 diluted primary goat polyclonal anti-Wnt-4 antibody (Santa Cruz Biotechnology) at 37°C for 60 minutes and washed three times for 5 minutes each with 1× TBS and then incubated with secondary rabbit anti-goat antibody for 30 minutes at room temperature. After three TBS washes, slides were incubated with 1:100 diluted streptavidin conjugated to horseradish peroxidase (Vector Laboratories, Inc.) for 60 minutes at room temperature. With AEC used as the chromogen, the rest of the immunohistochemistry steps were as just described.