Tumors grown in Cryab^−/− mice are less vascularized and show larger areas of apoptosis than tumors from wild-type (wt) mice. (A) Top panels, Immunohistochemistry of representative tumors grown in wild-type (left) or Cryab^−/− mice, showing CD31-positive vessels (brown) in viable areas of the tumors and large areas of necrosis/apoptosis (bar = 100 μM). Bottom panels, Immunofluorescent staining of representative tumors from each group showing apoptotic cells expressing cleaved caspase-3 (green) and proliferating cells expressing Ki-67 (red) in viable tumor areas (bar = 100 μm). (B) Quantification of mean cleaved caspase-3–positive area was done by analyzing 4× optical fields spanning all across a middle section of 4 different tumors per group using Easy Image Analysis. Bars represent mean values and SD of cleaved caspase-3−positive area (n = 4; mean ± SD; *P < .05). (C) The number of vessels/area was determined by counting the total number of CD31-positive and ASMA-positive vessels in each tumor by analyzing 10× optical fields spanning all across a middle section of 5 different tumors per group and normalizing relative to tumor area using Easy Image Analysis software. Results are given as percentage of vessels/area compared with wild-type mice, and bars represent mean values and SD (n = 5; mean ± SD; *P < .01). (D) Expression of αB-crystallin (red) was detected in a subset of CD31-positive vessels (green) in tumors grown in wild-type animals. Bar equals 20 μM.