PMC:2669177 / 37940-39151 JSONTXT

{"project":"2_test","denotations":[{"id":"19390689-17183666-97510257","span":{"begin":891,"end":893},"obj":"17183666"},{"id":"19390689-17183666-97510257","span":{"begin":891,"end":893},"obj":"17183666"},{"id":"19390689-16585269-97510258","span":{"begin":897,"end":899},"obj":"16585269"},{"id":"19390689-16585269-97510258","span":{"begin":897,"end":899},"obj":"16585269"},{"id":"19390689-17183666-97510257","span":{"begin":891,"end":893},"obj":"17183666"},{"id":"19390689-17183666-97510257","span":{"begin":891,"end":893},"obj":"17183666"},{"id":"19390689-16585269-97510258","span":{"begin":897,"end":899},"obj":"16585269"},{"id":"19390689-16585269-97510258","span":{"begin":897,"end":899},"obj":"16585269"}],"text":"Whole eyes were enucleated and fixed with phosphate-buffered saline containing 4% paraformaldehyde at 4°C overnight. With the exception of PI-2 eyes, the cornea and lens were removed and the eye was returned to fixative for an additional two hours. The eyes were cryoprotected by serial immersion in 15% and 30% (w/v) sucrose solutions for at least two hours each. Individual eyes were embedded in M1 embedding medium (Thermo Electron Corporation, PA) and frozen on dry ice; frozen sections (10 µm thickness) aligned with the vertical meridian were cut with a cryostat (Leica) and collected on precleaned Superfrost-plus® microscope slides (Fisher Scientific). The entire eye was sectioned, and every sixth section was collected, enabling examination of gene expression throughout the retina. For immunohistochemistry, all steps were carried out at room temperature as described previously [13], [47]. Staining controls included eyes from age-matched NMP transgenic and WT mice, and slides on which primary or secondary antibodies were omitted. Observation and imaging were performed using an epifluorescent microscope (AxiophotZeiss Ltd., Germany) and a spinning disk confocal microscope (BX62 Olympus, Japan)."}