PMC:1914394 / 54871-57608 JSONTXT

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Briefly, sections were incubated for 1 h at 37 °C with 75 μl of mixture solution consisting of 0.5 μl of terminal deoxynucleotide transferase, 1 μl of biotin-16-dUTP, 7.5 μl of CoCl2, 15 μl of 5× terminal deoxynucleotide transferase buffer, and 51 μl of distilled water. After three washes in 4× SSC buffer, sections were incubated with Alexa 488– or Alexa 568−streptavidin (1:1,000; Molecular Probes, http://probes.invitrogen.com) for 1 h at room temperature. Primary antibodies or labelled cells were visualized using donkey anti-mouse Alexa 488 or Alexa 568, donkey anti-rabbit Alexa 488 or Alexa 568, donkey anti-goat Alexa 488 or Alexa 568, and streptavidin Alexa 488 or Alexa 568 (1:1,000; Molecular Probes). Nuclei were counter-stained with 4,6-diamidino-2-phenyindole (DAPI; Sigma, http://www.sigmaaldrich.com). Labelled cells were visualized using a Zeiss (http://www.zeiss.com) Axioplan-2 microscope with Plan Neofluar objectives and images captured with a Zeiss AxionCam camera. For double-labelled samples, confocal images were obtained with a Zeiss LSM 5.0 laser scanning microscope.\nThe retina was separated into three bins by dividing the ventricular edge of the retina into equal parts and extending a line to the vitreal edge [2]. Bin 1 contains only cells that expressed Cre as progenitors; bin 3 is at central retina and contains cells derived from progenitors that did not express Cre. For cell counts or thickness measurement we used a region 0–100 μm peripheral to the boundary separating bins 1 and 2. Measurements were performed on an Axioplan-2 microscope using Axiovison software. Quantification of S-phase, M-phase, and apoptotic cells was performed on horizontal sections that included the optic nerve. Quantification of differentiated cell types was performed using horizontal sections at equal distances from the optic nerve. 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