VEGF expression was visualized in embryos from VEGF-LacZ mice by staining wholemounted E10.5 and E13.5 embryos for LacZ using the in situ β-galactosidase staining kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. As a negative control, whole embryos and cryosections from wild-type (WT) type mice were stained for LacZ and showed no positive staining in the eye (data not shown).
Embryos and lens explants were fixed overnight in 10% buffered formalin or 4% paraformaldehyde. The heads were placed in embedding molds, insuring proper orientation. For paraffin and cryosections, serial transverse sections covering the entire optic cup were cut and sections containing the largest area of the lens were selected for histology and immunochemistry. For histology, paraffin sections were stained with hematoxylin and periodic acid Schiff. For immunohistochemistry, rehydrated paraffin sections were boiled 30 minutes in citrate buffer pH 6 for antigen retrieval and pretreated with 1% H
2O
2 in methanol for 10 minutes to block endogenous peroxidase activity. Primary antibodies included rat anti-endomucin (1:500; provided by Dietmar Vestweber, Max-Planck-Institute, Bad Nauheim, Germany),
32 rabbit anti-mouse VEGFR2 T1014 (1:500; provided by Rolf Brekken, University of Texas Southwestern Medical Center, Dallas, TX)
33 34 and rabbit anti-mouse c-Maf (1:500, Santa Cruz Biotechnology, Santa Cruz, CA). Antibodies were visualized using avidin-biotin-peroxidase and DAB substrate (Vector ABC kit; Vector Laboratories, Burlingame, CA). For each experiment, a section was incubated with isotype-matched IgG as a negative control. After mounting, the sections were visualized and photographed using a microscope (Axioskop 2 FS mot; Carl Zeiss MicroImaging GmbH, Göttingen, Germany).
For fluorescent immunodetection, antisera included polyclonal antisera against αB-crystallin (1:1000; Serotec, Raleigh, NC), β-catenin (1:1000; Upstate, Billerica, MA), E-cadherin (1:500; Abcam, Cambridge, MA), N-cadherin (1:500; R&D Systems, Minneapolis, MN), MIP26 (1:500; kindly provided by Joseph Horwitz [Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA]), Phospho-histone H3 (Ser10; 1:40; Upstate), Prox1 (1:1000; Covance, Trenton, NJ) and p57kip2 (1:100; Abcam), followed by a rabbit or donkey raised Cy3-conjugated or FITC-conjugated secondary antibody (1:300; Jackson ImmunoResearch, West Grove, PA). Some sections were co-stained for F-actin using FITC-phalloidin (1:200; Molecular Probes, Carlsbad, CA). Cell nuclei were identified by DAPI labeling.