VEGF is expressed in the MMP-9−/− mice. (A–D) Bright-field (A,B) and dark-field (C,D) photographs of WT (A,C) and MMP-9−/− (B,D) sections of 1-week-old metatarsal growth plates hybridized with a Vegf antisense probe. Vegf is expressed in a subpopulation of hypertrophic chondrocytes (hc). (E,F) Immunostaining of tissue sections of 1-week-old WT (E) and MMP-9−/− (F) growth plates with VEGF antibody. VEGF protein is found in the ECM surrounding the hypertrophic chondrocytes (arrows). (G) Western blot of protein extracts from 2-week-old WT and MMP-9−/− growth plate HC probed with an anti-VEGF antibody showing that the major VEGF isoform expressed is VEGF164. Note the smaller forms of VEGF migrating around 28 kDa in both WT and MMP-9−/− HC with intact perichondrium (arrows). (H) Quantification by ELISA of VEGF protein secreted into the medium by cultured 2-week-old WT and MMP-9−/− growth plate ossification fronts. Bars, 200 μm (A–F).