斑马鱼肝脏组织油红O染色方法比较优化

Comparison and Optimization of Oil Red O Staining Methods in Liver Tissue of Zebrafish

目的通过设置不同染色步骤、条件,探索适合处理斑马鱼肝脏的冷冻切片油红O染色方法。方法取3月龄斑马鱼肝脏OCT样品,设置6、8、10、12μm四种不同厚度切片各两片,其他各组切片厚度均为8μm,采用不同的处理方法处理两张切片。染色后置于显微镜下观察视野内组织完整度、背景情况、橘红色脂滴的染色面积、位置以及深染的细胞和位置形态。结果以4%多聚甲醛固定、10%~30%蔗糖梯度脱水,切片厚度设置8~10μm,室温晾干1 h,甲醛(4%)固定10 min,油红O染色10 min,60%异丙醇分化2 s,ddH_(2)O浸洗浸泡5 s后,苏木素染色3 min,ddH_(2)O浸泡30 s去浮色,1%盐酸酒精分化2 s,流水冲洗2 min返蓝,甘油明胶封片为条件进行油红O染色的效果较好,斑马鱼肝组织组织完整、背景清晰,脂滴染色效果较好。结论优化的斑马鱼油红O染色方法可以较为清晰地显示肝脏脂滴,可辨别细胞核轮廓位置,进而对斑马鱼肝病模型中的脂肪变性情况进行定位与半定量的分析。

Objective To explore the suitable oil red O staining method for frozen section of zebrafish liver by setting different staining steps and conditions.Methods The OCT samples of 3-month-old zebrafish liver were taken.Four different thickness slices(6,8,10,12μm)were set,and the thickness of other slices was 8μm.After staining,the tissue integrity,background,staining area and location of orange lipid droplets,and deeply stained cells and location morphology were observed under a microscope.Results Zebrafish liver tissue was fixed with 4%paraformaldehyde,dehydrated with 10%-30%sucrose gradient,slice thickness was set at 8-10μm,dried at room temperature for 1 h,fixed with formaldehyde(4%)for 10 min,stained with oil red O for 10 min,differentiated with 60%isopropanol for 2 s,soaked in ddH_(2)O for 5 s,stained with hematoxylin for 3 min,soaked in ddH_(2)O for 30 s to remove the floating color,differentiated with 1%hydrochloric acid alcohol for 2 s,washed with water for 2 min to return to blue,and sealed with glycerin gelatin.The effect of oil red O staining was better,the liver tissue of zebrafish was complete,the background was clear,and the effect of lipid droplet staining was better.Conclusion The optimized zebrafish oil red O staining method can clearly show the liver lipid droplets and identify the position of the nuclear contour,and then locate and semi-quantitatively analyze the steatosis in the zebrafish liver disease model.