Finally, the sections were counterstained with 0.5% neutral red and dehydrated through a series of ethanol, mounted, and observed under a light microscope. Preparation of an Epitope-specific Polyclonal Anti-lumican Antibody To prepare the polyclonal antibody, a synthetic oligopeptide sequence (YYDYDIPLFMYGQISPNC) deduced from mouse lumican cDNA was conjugated to keyhole limpet hemocyanin (32). under a stereomicroscope as previously reported (29, 30). Neomycin ointment was topically applied to prevent bacterial infection. The animals were then killed at specific intervals of healing (1, 2, 4, or 8 h and 1, 2, 3, 5, 7, 14, 21, or 28 days). Each eye was removed, fixed in 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4) for 48 h, embedded in paraffin, and Bardoxolone methyl (RTA 402) processed for histology. In Situ Hybridization of Lumican mRNA Paraffin sections 5 hybridization with sense and antisense riboprobes of mouse lumican and mouse keratocan as previously reported (12, 31). Finally, the sections were counterstained with 0.5% neutral red and dehydrated through a series Mouse monoclonal to CD81.COB81 reacts with the CD81, a target for anti-proliferative antigen (TAPA-1) with 26 kDa MW, which ia a member of the TM4SF tetraspanin family. CD81 is broadly expressed on hemapoietic cells and enothelial and epithelial cells, but absent from erythrocytes and platelets as well as neutrophils. CD81 play role as a member of CD19/CD21/Leu-13 signal transdiction complex. It also is reported that anti-TAPA-1 induce protein tyrosine phosphorylation that is prevented by increased intercellular thiol levels of ethanol, mounted, and observed under a light microscope. Preparation of an Epitope-specific Polyclonal Anti-lumican Antibody To prepare the polyclonal antibody, a synthetic oligopeptide sequence (YYDYDIPLFMYGQISPNC) deduced from mouse lumican cDNA was conjugated to keyhole limpet hemocyanin (32). The polyclonal antibodies were raised in rabbits as explained previously (32). Anti-lumican antibodies were purified with an affinity column prepared by conjugating Bardoxolone methyl (RTA 402) the oligopeptide to Sulfolink? (Pierce) using the procedures recommended by the manufacturer. Western Blotting to Characterize the Anti-lumican Antibody Mouse corneal KSPGs and recombinant mouse lumican expressed in were prepared as explained previously (33), and the core proteins of the KSPGs were deglycosylated by treatment with wound healing model using cultured mouse eyes. Bardoxolone methyl (RTA 402) A Bardoxolone methyl (RTA 402) central corneal epithelial defect (2 mm in diameter) was produced in both eyes of 38 anesthetized wild-type mice under a stereomicroscope. Our preliminary immunohistochemical examination with a rat monoclonal anti-laminin antibody (X50, BIODESIGN International, Kennebunkport, ME) showed the presence of the uninterrupted epithelial basement membrane immediately after the epithelial scraping (data not shown). The animals were killed immediately after the epithelial dbridement. Each eyeball was enucleated and cultured in Dulbeccos altered Eagles medium (Life Technologies, Inc.) supplemented with 1.4% fetal calf serum and 50 role of lumican in corneal epithelial wound healing, we prepared lumican-null mice via gene-targeting techniques. The lumican gene-targeting construct contains 4.1 kb of 5-homology (cassette, 1.8 kb of 3-homology (cassette in the pBluescript vector. The cassette. The targeting vector was transfected into minigene; 381 base pairs), respectively. Northern hybridization, hybridization, and immunohistochemistry were used to determine the phenotypes of littermates using the procedures described above. Healing of Corneal Epithelial Defects in Lumican-deficient Mice Age-matched littermates were used as controls. Two-month-old = 22) and = 16) mice were anesthetized and subjected to 2-mm corneal epithelial dbridement as explained above. Our preliminary immunohistochemistry results showed the presence of the non-interrupted epithelial basement membrane immediately after epithelial scraping in both wound healing experiment explained above. RESULTS Characterization of Polyclonal Anti-Lumican Antibody We prepared epitope-specific anti-lumican antibody as explained under Methods and Methods. Western blot immune analysis was used to characterize an affinity-purified rabbit polyclonal antibody directed against the N-terminal oligopeptide of mouse lumican (YYDYDIPLFMYGQISPNC). This sequence is not found in keratocan or other members of the SLRP family (12, 27). Fig. 1 demonstrates that this antibodies reacted with recombinant lumican prepared from the expression clone of summarizes the strategy used to ablate the lumican gene in mice via gene-targeting techniques. A targeting construct made up of the human hypoxanthine phosphoribosyltransferase and herpes simplex virus thymidine kinase genes was prepared as explained under Materials and Methods. The genotypes of the lumican knockout mice were determined by PCR and Southern blot analysis (Fig. 2, and hybridization, and immunohistochemistry revealed no expression of lumican mRNA and the absence of lumican protein antigens in and gene. gene. In addition, a pMC-cassette was placed on the 3-end of the targeting vector. and hybridization (and hybridization.