摘要
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.
作者
张柏惠
杨晓倩
邹翔
曲中原
崔琳琳
郭守东
ZHANG Bai-hui;YANG Xiao-qian;ZOU Xiang;QU Zhong-yuan;CUI Lin-lin;GUO Shou-dong(School of Pharmacy,Harbin University of Commerce,Harbin 150076,China;Institute of Lipid Metabolism and Athero-sclerosis,School of Pharmacy,Weifang Medical College,Weifang 261053,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2021年第7期1329-1337,共9页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81770463,No.82070469,No.31300639)。
关键词
唾液酸
荧光标记
动脉粥样硬化
斑块着色
氧化应激
Sialic acids
Fluorescence labeling
Atherosclerosis
Plaque staining
Oxidative stress