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Updated: Apr 26, 2026
Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3
Published on: December 20, 2013
Bis(zinc-dipicolylamine), Zn-DPA, a new marker for apoptosis
Jacky M K Kwong1, Celia Hoang1, Reshil T Dukes1
1Jules Stein Eye Institute, University of California Los Angeles, Los Angeles, California, United States.
Purpose:
To characterize the labeling of apoptotic cells with a molecular probe of bis(zinc(II)-dipicolylamine) (Zn-DPA) conjugated with a fluorescent reporter in a rat model of retinal ganglion cell (RGC) degeneration induced by N-methyl-D-aspartate (NMDA).
Methods:
Adult Wistar rats were given unilateral intravitreal injections of 3 μL 40 mM neutralized NMDA and euthanized at 1, 2, 4, 24, and 48 hours. One hour before euthanasia, 3 μL Zn-DPA conjugated with fluorescein (Zn-DPA 480) was intravitreally injected. Prelabeling of RGC with retrograde fluorogold (FG), TUNEL, and immunohistochemistry with III β-tubulin and vimentin were performed.
Results:
Fluorescence labeling of Zn-DPA 480 was observed in the retinas from 1 hour up to 24 hours after NMDA injection, whereas the labeling was reduced at 48 hours postinjection. At both 4 and 24 hours postinjection, most Zn-DPA 480-positive cells in the RGC layer were labeled by FG and III β-tubulin. The number of TUNEL-positive cells increased from 4 to 24 hours. At 24 hours, 95.7% of Zn-DPA 480-positive cells were TUNEL positive, whereas 95.1% of TUNEL-positive cells were Zn-DPA 480 positive. The numbers of Zn-DPA 480-positive cells at 1 and 2 hours after NMDA injection were significantly higher than TUNEL.
Conclusions:
Our findings demonstrate that intravitreal injection of fluorescent Zn-DPA 480 labels retinal neurons undergoing apoptosis and that recognition of exposed phosphatidylserine appears earlier than detection of DNA fragmentation, indicating the potential of Zn-DPA as an imaging probe for tracking degenerating retinal neurons.
