Chick Embryo Ischemia Model Figure 5. Effect of ischemia on NO generation and peroxynitrite level in vitelline vascular tissue. (A) NO production by the ischemic vessels was measured using DAF-2DA fluorescent probe. Ischemic vessels were incubated for 10 min with 1 mM DAF-2DA dissolved in HBS containing 1 mM L- arginine. Images were taken under 4X objective of the microscope. Images are the representative of 3 individual experiments. (B) The green luminosity of the fluorescent images was analyzed using Adobe Photoshop 7.0. Luminosity of the images was directly proportional to the level of NO produced thus plotted as index of NO production. **P,0.001 versus non-ischemic. (n = 3) (C) Peroxy nitrite generation was measured using DHR123 fluorescent probe. Tissues were incubated with DHR123 for 2 h followed by homogenization and centrifugation for 5 min at 6500 g. Supernatant was read at excitation/ emission of 500/536 nm and the values were normalized with the weight of the tissues and plotted. *P = 0.03 versus non-ischemic. (n = 5). doi:10.1371/journal.pone.0010524.g005 Figure 6. Effect of ischemia on GSH level in vitelline vascular tissue. (A) Level of GSH was measured using CMFDA fluorescent probe. The ischemic vessels were incubated with CMFDA probe for 30 min. Next, the images were acquired with DP71 CCD camera. Images are the representative of 5individual 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 GSH in the vessels. **P,0.001 versus non-ischemic. (n = 5) (C) Ischemic tissues were probed with CMFDA for 30 min. Tissues were then homogenized and centrifuged at 6500 g for 5 min. The supernatants were then read at excitation/emission of 485/515 nm. Values obtained were normalized with the weight of the tissues. **P,0.001 versus non-ischemic. (n = 5). doi:10.1371/journal.pone.0010524.g006 PLoS ONE | www.plosone.org 5 May 2010 | Volume 5 | Issue 5 | e10524