Acetyl asp glu val asp 7 amido 4 methylcoumarin

Apoptosis were detected with TUNEL assay. Embryos were harvested at the indicated stages, fixed in 4% PFA, incubated in 1μg/ml Proteinase K solution to solubilize tissues and rinsed twice with PBS-Tween. Apoptotic cells were labeled using ApopTag® Red In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA) following the manufacture’s instruction. Fluorescent images were taken with a LSM 710 confocal microscope from Carl Zeiss. To detect the activity of caspase 3 in zebrafish, embryos were collected at 60 hpf and homogenated in ice-cold RIPA buffer. Then, the homogenate was mixed with an equal volume of 0.39 mM of Acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Sigma-Aldrich, Saint Louis, MO, USA), a fluorogenic substrate for caspase 3. Quantification of caspase 3 activity was performed by fluorescent detection of free AMC (also known as 7-amino-4-methylcoumarin) as described previously [43 (link)].

Calcium ionophore (A23187), benzamidine hydrochloride, N-acetyl-Leu-Glu-His-Asp trifluoro methylcoumarin (AC-LEHD-FMC), acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (AC-DEVD-AMC), sodium orthovanadate, dimethyl sulfoxide (DMSO), fluorescein isothiocyanate (FITC)-labeled annexin V, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetylester (CM-H2DCFDA), CHAPS, rhodamine 123, leupeptin hydrochoride, N-(2-Hydroxyethyl) piperazine-N′-ethanesulfonic acid (HEPES), fura-2/AM, monoclonal anti-phosphotyrosine antibody, acridine orange 10-nonyl bromide (NAO) and dithiothreitol (DTT) were from Sigma Chemicals, St. Louis (USA). Monoclonal anti-cytochrome c antibody and anti-β-actin were from Epitomics Burlingame, CA (USA). Anti-Caspase-3 antibody was from Santa Cruz Biotechnology, Inc. Texas (USA). Collagen type-I was from Chrono-log Corporation, Pennsylvania (USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 1, 1-diphenyl-2-picrylhdrazyl (DPPH) were from HiMedia Laboratories, Mumbai (India). Lactate dehydrogenase (LDH) kit was from AGAPPE diagnostics Ltd., Kerala (India). γ-glutamyl p-nitroanilide and glycylglycine were from Sisco Research laboratories Pvt Ltd., Mumbai (India). All other reagents were of analytical grade.