Chick Embryo Ischemia Model Figure 14. Effect of ischemic vascular driven secondary ischemia on caprine thoracic goat heart strip. (A) Caprine thoracic goat hearts were obtained from government authorized slaughter houses. Thin section of the hearts were made using surgical blade and placed on the top of ischemic vascular bed of chick embryo and incubated for 2 h. Next, the heart strips were homogenized in HBS containing protease inhibitor and centrifuged at 10,000 g. The CK-MB activity was then measured in the supernatant and the values obtained were plotted after normalizing with the weight of the tissues. Ischemia induced the CK-MB activity of the heart muscle. **P,0.001 versus non-ischemic. (n = 3) (B) Heart strips were treated under ischemia and fixed with 10% formalin. Next, the strips were embedded in paraffin wax and sectioned. The thin sections were stained with hematoxin-eosin stain and imaged with Nikon Cool Pix camera adapted to a stereo microscope. Ischemia induced edema and an elevated intra- mascular gaps as showed by the black arrows. Images are the representative of 5 individual experiments. doi:10.1371/journal.pone.0010524.g014 minutes. The fluorescence of the supernatant was measured on Varian Carry Eclipse Fluorescence Spectrophotometer at 553 nm with an excitation wavelength of 515 nm. The level of fluores- cence was taken as an index of lipid peroxidation both in tissue and cellular systems. Cell viability assay using trypan blue Viability experiment was performed following the protocol as mentioned earlier [55]. In brief, EAhy926 cells were grown to about 60% confluence in collagen coated cover slips. The cells were then subjected to ischemia treatment. Media containing trypan blue (0.4 mg/ml) was added to the cells and incubated for another 15 min. After incubation, the media was removed and PBS was added to all the wells. Random images were acquired using Nikon Cool Pix camera adapted to a Motic inverted microscope and the numbers of cells having a blue colored nucleus were counted from the images. Apoptosis measurement using annexin V-FITC apoptosis detection kit EAhy926 cells were grown overnight on collagen coated cover slips and treated under partial ischemia using the ischemic vascular bed of chick embryo. Next, treated cells were processed using the protocol as supplied by the manufacturer (Merck, Calbiochem, EMD Chemicals Inc., Darmstadt, Germany). Fluorescence images of the cells were taken and the number of annexin V-FITC positive cells were counted per field. Angiogenesis pattern of the vascular bed of chick embryo adjacent to the ischemic area HIF-1a is a known inducer of angiogenesis [5]. In the present study, we wanted to evaluate the effect of secreted HIF-1a from the ischemic vessels on angiogenesis pattern of the adjacent vessels which are non ischemic. To study the same, we followed the angiogenesis pattern of the vessels, which is just adjacent to the ischemic vessels. The images of the vessels at different time interval were acquired during the period of ischemia using a Nikon Cool Pix camera adapted with a stereo microscope. Images were then analyzed using Adobe Photoshop version 7.0 and Angioquant software [56]. Morphology counting of the cells under ischemia EAhy926 and H9C2 cells were incubated on the top of the ischemic vascular bed. After 2 h incubation, cells were imaged with Nikon Cool Pix camera adapted to Motic inverted microscope. For reversible experiment, cells were first imaged and incubated under ischemia. After treatment, cells were again imaged and kept on the top of a normal vascular bed for another 2 h. After 2 h incubation, cells were imaged. For RL, NAC and TRZ based recovery experiments, cells were pretreated with RL, NAC and TRZ for 15 min placed on the top of ischemic vessels treated with RL, NAC and TRZ respectively. RNA isolation and quantitative RT-PCR Total RNA was isolated from ischemia treated EC and ischemic tissue itself using spin prep kit (Medox Inc) and quantified and normalized according to the band intensities. cDNA synthesis was performed on 200 ng of RNA using mulv reverse transcriptase (New- England Biolabs) and PCR was performed using 50 ng cDNA. The reactions were carried out for 30 cycles at 92uC denaturation for 1 min, 57uC annealing for 2 min and 72uC elongation for 3 min. The forward primer sequence is 5-AGATCTCGGCGAAGTAAA- GAGTCTGAAGT-3 and the reverse primer sequence is 5- AGCATCCTGTACTGTCCTGTGGTGACTTGT-3 for human HIF-1a while forward primer sequence is 5- ACTTCAGCAGACT- CAGAC-3 and the reverse primer sequence is 5-GTT- CCAATGTTCCTTTTC-3 for avian HIF-1a, which amplifies the human and avian HIF-1a sequence (3376 bp–3691 bp) with a PLoS ONE | www.plosone.org 13 May 2010 | Volume 5 | Issue 5 | e10524