Chick Embryo Ischemia Model Figure 12. RL driven recovery of ischemia treated EC activity. (A) Number of round cells per field were counted from the bright field images. Data were normalized by counting the total number of cells and presenting as percentage of round cells per filed. RL, NAC and TRZ attenuated ischemia driven rounding up of the cells. No effect of RL, NAC and TRZ were observed under non-ischemic condition. **P,0.001 versus non-ischemic and yyP,0.001 versus ischemia. (n = 7) (B) Green luminosity from the images was measured using Adobe Photoshop version 7.0. Fluorescence intensities of 40 cells from 5 different sets of experiments were calculated and plotted. **P,0.001 versus non-ischemic and yyP,0.001 versus ischemia. (n = 5). (C) Number of round cells was counted per field from the bright field images. Data were normalized by counting the total number of cells in each field and plotting as percentage of round cells per field. **P,0.001 versus non-ischemic and yyP,0.001 versus ischemic. (n = 7). doi:10.1371/journal.pone.0010524.g012 embryo body and another vitelline system extending into the egg sac, which helps the embryo to get nutrients from the yolk and diffused oxygen from the air. Blocking the circulation of any of the vitelline artery can interfere with nutrient and oxygen diffusion to the blood thus can create a partial ischemia. In the present study, ischemia was created in the vascular bed of chick embryo by using a modified protocol as mentioned by Vos et al., 2003 where they studied the hemodynamics due to blockade in right vitelline vein [51]. 96 h grown chick embryos were broke open in a sterile glass bowls (Fig. 1A). Only embryos that showed no bleeding or deformities were selected. The material was subdivided into artery ligated embryos and control embryos. In the ligation group, a small incision in the yolk sac membrane was made. Subsequently, the right vitelline artery was blocked using a surgical suture (Fig. 1B). Cessation of blood flow proximal to the ligation was confirmed under stereo microscopic surveillance. The sutures were made up of 0.1 mm thick bio-friendly and biodegradable surgical thread. All experiments were performed ex vivo. During the experiments, all eggs were placed on a thermo element of 37uC/humidity and covered with a sterile glass lead. Preparation of the tissue samples for measuring HIF-1a and different ROS parameters After partial ischemia, vascular bed of the chick embryo was surgically incised out using a sterile surgical scissor. For mapping lipid peroxidation, the whole vascular bed along with the embryo was taken off from the yolk sac and placed in Petri plate containing PBS buffer. Next, the vascular bed was dissected out from the embryo into six equal sections. At the end of the dissection, the embryo left untouched while the whole vascular bed were sectioned to six equal portions. The left non-ligated portion was taken as our control tissue. Tissues were then washed 3–4 times with HBS in order to wash out all the yolk material and the blood from the vessels. Care was taken to avoid any damage to the vascular bed of chick embryo. Next, respective tissue samples were processed according to the experimental need. Treatment of different cells under ischemia using the ischemic vascular bed of chick embryo Work of different groups showed that mammalian cells can be grown easily using the hen’s yolk as growth media [52,53]. PLoS ONE | www.plosone.org 11 May 2010 | Volume 5 | Issue 5 | e10524