Verification of collagen transcripts that were upregulated the most in the late phase of conjunctival wound healing by qPCR. (A) Real-time PCR analyses of the 10 most highly expressed collagen genes in mouse conjunctival tissues harvested 2, 7, 14 and 21 days after experimental surgery. Data are calculated as fold-change relative to paired unoperated controls. The mean fold-change of five groups, each consisting of operated tissues pooled from three mice, for each time point is indicated (n=5). *P<0.05 for fold-change compared with all other time points; ^δP<0.05 for fold-change on day 7 compared with both days 14 and 21; ^ψP<0.05 for fold-change on day 7 compared with day 21 only. (B) Immunoblot analyses of COL8A1, COL11A1 and COL1A1 in mouse conjunctival tissues harvested on day 2 and day 7 post-experimental surgery. Three paired groups for each time point are shown (n=3). Op, operated tissues pooled from five independent eyes per group; C, paired untreated controls pooled similarly. Fold-change in expression in operated relative to control tissues, both normalized to GAPDH, and the associated P values corrected by Bonferroni adjustment, where significant, are shown below the respective immunoblot.

Immunolocalization of COL8A1, COL11A1 and COL1A1 in the day 7-operated mouse conjunctival tissue. (A) Co-immunolabelling of COL8A1 (red), COL11A1 (green) and COL1A1 (magenta). Upper panels, insets (i) show magnified images of the boxed areas in the first and third images, with yellow and white arrowheads indicating high COL8A1/low COL1A1 and low COL8A1/high COL1A1 co-immunolabellings, respectively; inset (ii) left, second image, shows a magnified image of the boxed area co-immunolabelled for COL8A1 and COL11A1; inset (ii) right, second image, shows a magnified image of the boxed area co-immunolabelled for COL11A1 and COL1A1; inset (ii), fourth image, shows a magnified overlay image of the boxed area co-immunolabelled for all three collagens. None of the insets includes DAPI staining. Lower panels, a section from the same eye incubated with only the secondary antibodies used in the upper panel showed minimum non-specific staining in the bleb area in comparison with non-specific staining, which was observable in the choroid (arrow). S, sclera. (B) Co-immunolocalization of COL8A1 (green) and COL1A1 (magenta) with ACTA2 (red). Yellow and white arrowheads in insets indicate COL8A1 and COL1A1 co-immunolabelling patterns, as indicated in A. Co-immunolabelling of ACTA2 with COL8A1 (left inset, second image) or COL1A1 (right inset, second image) appears yellow or blue, respectively. Co-immunolabelling of COL8A1 and COL1A1 appears white (right inset, third image). None of the insets includes DAPI staining. (C) Co-immunolocalization of COL11A1 (green) with F4/80+ cells (red). Inset, fourth image, shows a magnified overlay image of the boxed area co-immunolabelled for COL11A1 and F4/80. All pictures were captured by confocal microscopy. Scale bars: 100 μm.

Induction of top collagen genes by TGFβ2 in primary mouse conjunctival fibroblasts. (A) Real-time PCR analyses of collagen genes in fibroblasts stimulated for 72 h with TGFβ2. Data shown are representative of three independent experiments, and show mean fold-change±s.d. relative to untreated controls. *P<0.05 for fold-change in treated versus control. (B) Immunoblot analyses of COL8A1, COL11A1 and COL1A1 in mouse fibroblasts treated as indicated for 72 h. Three independent sets of experiments are shown. Fold-change in expression in TGFβ2-treated relative to control cells, both normalized to GAPDH, and P value (corrected by Bonferroni adjustment) where significant, are shown in the densitometric analyses below the immunoblot.

Induction of top collagen genes by TGFβ2 in primary human conjunctival fibroblasts and in individuals with surgical failure. (A) Real-time PCR analyses of COL8A1, COL11A1, COL8A2 and COL1A1 in primary human conjunctival fibroblasts stimulated with TGFβ2 for 72 h. Data shown are representative of three independent experiments, and calculated as mean fold-change±s.d. relative to untreated controls. *P<0.05 for fold-change in treated versus control. (B) Immunoblot analyses of COL8A1, COL11A1 and COL1A1 in human fibroblasts treated as indicated for 72 h. Three independent sets of experiments are shown. Fold-change in expression in TGFβ2-treated relative to control cells, both normalized to GAPDH, and P values (corrected by Bonferroni adjustment) where significant, are shown in the densitometric analyses below the immunoblot. (C) Real-time PCR analyses of COL8A1 and COL1A1 in human conjunctival tissues from individuals requiring repeat surgeries (n=15) relative to those with no prior GFS (n=20). Data for COL8A1 in tissues of individuals with no prior surgeries was only available from 15 patients as five samples did not produce detectable values. Data shown are calculated as fold-change in expression in tissues of repeat surgery patients relative to that from patients with no prior surgery. Significant fold-changes between the two groups of patients and the associated P values (*) are indicated. Each symbol represents one patient.