Figure 5.

Systemically injected fluorescein isothiocyanate (FITC)-conjugated annexin V labels cells undergoing apoptosis in the developing retina. A, Mouse pups were injected with either FITC-annexin V or FITC-dextran 4 hours postexposure to either isoflurane or air. Immunohistochemistry for activated caspase-3 was performed on retinal sections 1 hour postinjection. Representative sections imaged at 40× are depicted. Scale bar, 50 μm. Green annexin V–positive cells and red activated caspase-3 (casp-3)-positive cells are visible within the inner nuclear layer (INL) of air-exposed and isoflurane (Iso)-exposed animals. Merged images demonstrate colocalization of FITC-annexin V with activated caspase-3 in the INL of both air-exposed and isofluane-exposed cohorts (arrowheads). B, Representative images of cells fluorescent for both activated caspase-3 and annexin V magnified at 100× are demonstrated. Merged images demonstrate colocalization of fluorescence. All FITC-annexin V–positive cells demonstrated colocalized fluorescence with activated caspase-3. Activated caspase-3-positive cells lacking annexin V fluorescence are also shown. Scale bar, 10 μm. C, Quantification of FITC-labeled cells after annexin V or dextran injection in air-exposed and isoflurane (Iso)-exposed cohorts is demonstrated. Values are expressed as means + SD. N = 5–6 animals per cohort. †P = 0.002 versus air-exposed cohort. D, Percentage of activated caspase-3-positive cells that demonstrated FITC-annexin V fluorescence (white bars) and that lacked FITC fluorescence (activated caspase-3 only; black bars) in both air-exposed and isoflurane-exposed cohorts is demonstrated. Values are expressed as means + SD. N = 5–6 animals per cohort. †P = 0.002 versus air-exposed cohort. GCL = ganglion cell layer; ONL = outer nuclear layer; DAPI = 4′,6-dimidino-2-phenylindole.