明视野显微镜下特异性区分斑马鱼视网膜细胞核的方法

摘要

目的：结合 JB-4塑胶树脂包埋和福尔根染色寻找一个最佳能够敏感且特异染色斑马鱼视网膜细胞核的方法。方法分析 JB-4切片厚度、盐酸水解程度及 Schiff 反应条件对细胞核染色的影响；观察不同切片厚度对细胞核可分辨度的影响；比较福尔根染色与亚甲天蓝 II 及核固红染色对细胞核观察的影响。结果盐酸浓度越高、浸润时间越长，染色程度越强；盐酸浓度越低、浸润时间越短，染色程度越弱。低浓度的盐酸可以通过延长浸润时间而提高染色强度。染色强度随着 Schiff 浸润时间的延长而增加，2 h 达到最强染色。在2μm 组织切片中单个细胞核比在3μm组织切片、4μm 组织切片及6μm 组织切片中显示出更清晰的核边界。与亚甲天蓝 II 和核固红染色相比，福尔根染色特异性和稳定性更强。结论通过本实验结果分析发现了一个最佳能够敏感且特异染色斑马鱼视网膜细胞核的方法，这个简单可靠的染色方法可以用于细胞计数。%Objective To optimize a protocol that produces sensitive and specific nuclear staining of the zebrafish retina combining JB-4 plastic embedding and Feulgen staining.Methods The effects of JB-4 section thickness,extent of HCl hydrolysis,and Schiff reaction conditions on nuclear staining were analyzed.The effects of section thickness on nuclear visualization were observed.Feulgen staining with methylene blue-Azure II and nuclear fast red staining on nu-clear visualization were compared.Results The results showed that higher HCl concentrations and longer incubation produced stronger staining than lower HCl concentrations and shorter incubation time.And lower HCl concentrations can be combined with longer incubation to achieve similar staining results.We found that staining became stronger with longer Schiff incubation but reached the maximum after 2 hour.Individual nuclei showed more clear boundaries in 2 μm sections than in 3 μm,4 μm,and 6 μm sections.Feulgen staining provided a more specific and stable staining than methylene blue-azure II and nuclear fast red staining.Conclusion We optimize a protocol that produces sensitive and specific nuclear staining of the zebrafish retina.This simple and robust method may be utilized for cell quantification.