Objective lt;brgt;To investigate the feasibility of labeling iris pigment epithelial（IPE）cells of rabbits with 5（and 6）carboxyfluorescein diacetate succinimidyl ester（CFSE）. lt;brgt; lt;brgt;Methods lt;brgt;Enzyme-assisted microdissection was used to isolate the cultured rabbitprime;s IPE cells.The third or forth subcultured IPE cells were incubated with 2.5，5，10，20，and 40 mu;mol/L of CFSE for 1，5，and10min respectively.The fluorescence intensity was detected by flow cytometry，and the leakage of CFSE and its dyeing were observed by fluorescence antibody labeling. lt;brgt;Results lt;brgt;Incubation with 20 mu;mol/L CFSE under 37℃for1minute was the most optimal condition for IPE cells labeling.The coloration of IPE cells stained by CFSE lasted 4 weeks.There was no leakage of dye from labeled rabbit IPE cells to non-labeled human IPE cells in mixed culture process. lt;brgt; lt;brgt;Conclusion lt;brgt;With the advantages of high rate of dyeing，long time of tracing，safety and convenience，CFSE can be used as a new method to label the rabbitprime;s IPE cells. lt;brgt; lt;brgt;(Chin J Ocul Fundus Dis, 2006, 22: 261-264)
Objective To evaluate the feasibility of imaging the rat cardiac conduction system (CCS) using transaortic antegrade perfusion of Alexa Fluor 633-labeled antibodies targeting hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4) and connexin (Cx). The study also sought to optimize antibody dosage, perfusion duration, and assess the photostability of the dye. Methods Ex vivo rat heart model with transaortic antegrade perfusion was established using 33 male SPF-grade Sprague-Dawley (SD) rats. Primary and secondary antibody solutions were sequentially perfused in an antegrade manner. After perfusion for predetermined durations, the atrioventricular junction was observed, and the fluorescence intensity of the corresponding area was recorded. Five dose-gradient groups (n=3 rats/group), five perfusion time-gradient groups (n=3 rats/group), and ten continuous LED light exposure time-gradient groups (using 3 rats prepared with a fixed dose and time) were established to observe and record regional fluorescence intensity. Standard immunofluorescence staining was performed on both paraffin and frozen sections for comparative histological analysis. Results A region of aggregated red fluorescent signal was observed in the atrioventricular junction. Following semi-quantitative fluorescence intensity analysis of HCN4/Cx43 and validation through comparative histology, this structure was identified as the atrioventricular node (AVN) region. The AVN-to-background fluorescence intensity ratio showed no statistically significant differences among groups with increasing antibody dosage (P>0.05). The ratio increased with longer antibody perfusion times. Furthermore, no statistically significant differences in the ratio were observed among groups with extended light exposure (P>0.05). Conclusion Transaortic antegrade perfusion of fluorescently labeled antibodies can successfully image the AVN within the CCS of ex vivo rat hearts. Increasing the antibody dosage does not significantly improve the AVN imaging effect. Longer antibody perfusion time results in better imaging quality of the AVN. The fluorescent dye maintains sufficient visualization of the AVN even after prolonged (8 h) exposure to light.