Chick Embryo Ischemia Model Figure 3. Effect of ischemia on superoxide generation in vitelline vascular tissue. (A) Vessels were probed with NBT for 1 h. Next, the images of the vessels having the purple formazen crystal were acquired with Nikon Cool Pix camera adapted to a stereo microscope. Images are the representative of 5 individual experiments. (B) The number of purple formazen crystals in the vessels was counted manually. Data were normalized by taking the area of the vessels and plotting as number of purple spots/unit area. **P,0.001 versus non-ischemic. (n = 5) (C) Ischemic vessels were incubated with NBT for 1 h followed by homogenization of the tissue and centrifugation of the suspension at 6500 g for 10 min. The supernatant was then read at 560 nm. Data were normalized with the weight of the tissue. **P,0.001 versus non-ischemic. (n = 3). doi:10.1371/journal.pone.0010524.g003 Figure 4. Effect of ischemia on hydrogen peroxide generation in vitelline vascular tissue. (A) Ischemia treated vessels were probed with amplex red for 30 min. Fluorescent and bright filed images were acquired with DP71 CCD camera adapted to Olympus IX71 microscope. Images are the representative of 5 individual experiments. (B) The fluorescent intensities of the images were calculated using Adobe Photoshop 7.0 and plotted. Fluorescent intensities were considered as directly proportional to the level of hydrogen peroxide produced. **P,0.001 versus non-ischemic. (n = 5) (C) Ischemic tissues were probed with amplex red for 30 min followed by homogenization and centrifugation at 6500 g for 10 min. The supernatant was then read at excitation/emission of 570/585 nm. Values obtained were normalized with the weight of the tissues. *P = 0.023 versus non-ischemic. (n = 3). doi:10.1371/journal.pone.0010524.g004 PLoS ONE | www.plosone.org 4 May 2010 | Volume 5 | Issue 5 | e10524