Triplex IHC was performed for α-SMA (myofibroblast marker, red), vimentin (mesenchymal cell marker, at a concentration where all corneal fibroblasts and myofibroblasts, but only rare keratocytes were positive, yellow),
9,10 and keratocan (marker for keratocytes, green), using previously described methods
6 and primary antibodies confirmed by Western blotting and IHC to recognize rabbit antigens or isotypic non-specific control antibodies (ThermoFisher Scientific, Waltham, MA, USA), and previously described secondary fluorescent tagged antibodies (
Table).
6 IHC was also performed for TGF-β1 and collagen type IV with 4′,6-diamidino-2-phenylindole staining of nuclei using previously characterized primary and secondary antibodies (
Table).
6 The TGF-β1 antibody used in this study does not bind TGF-β2 or TGF-β3.
6 The collagen type IV antibody (cat. AB769; Millipore, Temecula, CA, USA) used in this study was generated against purified human and bovine collagen type IV and affinity purified with human and bovine collagen type IV crosslinked to agarose and cross-absorbed by the manufacturer with human and bovine collagens type I, II, III, V, and VI to eliminate cross-reactivity. This collagen type IV antibody was shown previously to bind rabbit collagen IV in IHC
7 and binds the α1/α2 chains but not the α3 to α6 chains. Winston Kao, Ph.D., graciously provided the keratocyte-specific keratocan antibody raised against peptide H2N-LRLDGNEIKPPIPIDLVAC-OH.
6,7 Images were obtained at total magnification ×200 on a Leica DM6B upright microscope equipped with an automated stage and Leica 7000 T camera using the LASX software (Leica Microsystems, GmbH, Wetzlar, Germany).