一种生物膜表面唾液酸的染色和定量方法

A method for staining and quantification of sialic acids anchored on sur⁃face of biomembrane

目的:建立一种细胞、组织和器官生物膜表面唾液酸的染色及定量方法。方法:设计平行实验,确定最佳染色条件。将接种在盖玻片上的细胞用PBS洗涤后,加高碘酸钠(NaIO4,1 mmol/L)于4℃轻度选择性氧化生物膜表面的唾液酸在C-7位成醛;随后,加入甘油工作液终止氧化,再用冷的PBS清洗;再加入荧光素-5-氨基硫脲(FTSC,100μmol/L)荧光染料与生成的醛基避光反应,并采用PBS洗涤;最后,采用DAPI染核并采用PBS-T清洗,甘油封片、拍照,采用图像处理软件量化分析。组织切片经多聚甲醛固定后,按照上述优化后的步骤染色。结果:平行实验结果显示,FTSC染色40 min细胞表面唾液酸的成像效果最佳;3种不同的细胞经唾液酸染色后均呈现强的绿色荧光,经100μmol/L H_(2)O_(2)处理24 h后,荧光强度显著降低(P<0.01);该方法可原位检测小鼠组织切片和动脉内壁的唾液酸水平。假阳性结果可通过纳入仅采用FTSC处理组排除或扣除背景。结论:该方法可对不同细胞、组织和器官表面的唾液酸进行原位染色及定量分析。

AIM:To establish a method for staining and quantification of sialic acids on the biomembrane.METHODS:Parallel experiments were designed to optimize the staining conditions.Cells were seeded on glass slides in 6-well plates.After attachment or treatment,the cells were gently washed with cold PBS three times,and 1.0 mL sodium metaperiodate(NaIO4)solution(1 mmol/L)was added to each well and maintained at 4℃ for 20 min to mildly oxidize sialic acids anchored on biomembrane,inducing the production of aldehydes at C-7.This mild oxidation was quenched with 1.0 mL glycerol(1 mmol/L),and the cells were washed three times with cold PBS.Fluorescent dye fluorescein-5-thiosemicarbazide(FTSC,100μmol/L)in PBS(pH 7.4)was added to react with the aldehydes at 37℃for 40 min in dark.Subsequently,the cells were gently washed with PBS three times and were stained with DAPI for 5 min.Finally,slides were mounted and visualized using microscope.Images were recorded and analyzed using the ZEN imaging soft-ware.The tissue sections were fixed in 4% paraformaldehyde,stained,and quantified as described above.RESULTS:The optimized FTSC treatment time was 40 min.After staining,sialic acids showed strong green fluorescence as demon-strated in 3 kinds of cells.However,the green fluorescence was significantly decreased after the cells were treated with 100μmol/L H_(2)O_(2) for 24 h(P<0.01).This method was used to stain and quantify the levels of sialic acids in different ani-mal tissue sections.Furthermore,the false-positive result was avoided via setting FTSC-only treatment group.CONCLUSION:This method can be used for in situ staining and quantitative analysis of sialic acids anchored on the biomembrane of different cells,tissues,and organs.