Heparanase expression in vivo upon DTH induction. (A, B) Endogenous heparanase: 5 days after sensitization, left ear (B) of female BALB/c mice (n = 4) was treated with oxazolone and the right ear (A) with vehicle alone. Ear tissues were harvested 24 hours after challenge and processed for immunohistochemical analysis of heparanase expression (reddish staining). Vascular structures were recognized as luminal or slit-like structures that occasionally contained blood cells and were delineated by flattened endothelial cells. This experiment was repeated 3 times, and a similar immunostaining pattern was obtained with 2 different antiheparanase antibodies. Representative microphotographs are shown. (A) Nonchallenged ear: capillary endothelial cells in the ear skin dermis are negative for heparanase staining (magnification × 1000). (B) Oxazolone-challenged ear: heparanase-expressing capillary endothelial cells are easily detected (× 1000). Control sections stained using secondary antibody alone showed no staining. (C, D) When DTH reaction was elicited in hpa-tg mice, positive staining was detected in capillary endothelium both prior to (C) and after (D) the challenge. Images were captured with a Zeiss Axioskop 50 microscope (Zeiss, Oberkochen, Germany) equipped with 100 ×/1.30 oil objective or 20 ×/0.50 objective lenses. Images were captured with a Kodak DC290 digital camera (Kodak, Rochester, NY).