Annexin 5 fluorescein isothiocyanate fitc

The apoptotic cells were labeled in vivo with an intravitreal injection of 2 μg/10 µl of annexin V-fluorescein isothiocyanate (FITC; Santa Cruz Biotechnology, Inc., Dallas, TX) 24 h or 7 days after the rtPA injection in the healthy rats and 18 h after the NMDA ± rtPA injection in the excitotoxicity model. The animals were euthanized (an overdose 150 mg/kg intraperitoneal pentobarbital) 1 h after the annexin V-FITC injection, and the eyes were removed immediately and fixed for 15 min in 4% paraformaldehyde (PAF) at room temperature. Then the retinas were flatmounted, fixed in acetone at −20 °C for 10 min, and permeabilized with PBS/0.3% Triton for 20 min. 4',6-diamidino-2-phenylindole (DAPI) staining (1:5,000; Sigma-Aldrich, St. Louis, MO) was performed for 10 min. After rinsing with PBS, the retinas were finally flatmounted between a slide and a coverslip using mounting medium (Vectashield©, Vector Laboratories Inc., Burlingame, CA). They were visualized on a fluorescence microscope (BX51, Olympus, Rungis, France). Twelve photographs were taken following a cross pattern centered on the optic nerve. Annexin V-FITC-positive cells were counted using ImageJ software (Version 1.47p, National Institutes of Health, Bethesda, MD).

U118 and U138 human glioma cell lines were purchased from the Health Science Research Resources Bank (Osaka, Japan). GC, all-trans RA, dimethylformamide (DMF), dimethylsulfoxide (DMSO), dialysis membranes (molecular weight cut-off, 12,000 g/mol) and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluorescein isothiocyanate (FITC)-Annexin V was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The present study was approved by the Institutional Review Board and Ethics Committee of the Nanjing University (Jiangsu, China).

Ce6 was obtained from Frontier Scientific Inc. (Logan, UT, USA). 2′,7′-dichlorofluorescein diacetate (DCFH-DA), MTT, propidium iodide (PI), mercaptosuccinic acid (MS), and GSH were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Fluorescein isothiocyanate (FITC)-Annexin V was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Cell culture materials were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The total GSH detection kit, GPx activity kit, and GR activity kit were obtained from Enzo Life Sciences (Farmingdale, NY, USA).