苏丹黑B降低大鼠肠道自发荧光及其在免疫荧光染色中的应用

李勇; 周立; 张阳; 刘震; 姚念; 申凤鸽; 王志勇

doi:10.16098/j.issn.0529-1356.2020.03.024

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解剖学报 ›› 2020, Vol. 51 ›› Issue (3) : 450-455. DOI: 10.16098/j.issn.0529-1356.2020.03.024

技术方法

苏丹黑B降低大鼠肠道自发荧光及其在免疫荧光染色中的应用

李勇¹ 周立¹ 张阳¹ 刘震² 姚念²申凤鸽¹王志勇^1*

作者信息 +

Sudan black B reducing the intestinal autofluorescence of rats and its application in immunofluorescence

LI Yong¹ ZHOU Li¹ ZHANG Yang¹ LIU Zhen² YAO Nian² SHEN Feng-ge¹ WANG Zhi-yong^1*

Author information +

文章历史 +

摘要

目的探讨降低大鼠肠道组织自发荧光的简便可行方法。方法取正常SD大鼠（n=5）肠道做冷冻切片，荧光显微镜下观察自发荧光（AF），检测苏丹黑B（SBB）对AF的影响及其在肠道免疫荧光（IF）染色中的应用。结果大鼠各肠段组织在FITC和TRITC通道存在大量明显的AF，且不受血清封闭的影响；应用0.3% SBB处理组织10 min，可显著降低AF；IF染色加SBB处理后的肠道可见AF显著降低，目的蛋白的荧光表达不受影响。结论 0.3% SBB染色10 min能显著降低大鼠肠道组织的AF，使IF特异性染色更加鲜明。

Abstract

Objective To develop a feasible method to reduce the rat intestinal autofluorescence (AF). Methods Five rats were sacrificed. After cryostat section, intestinal AF was investigated by immunofluorescence (IF) before and after Sudan black B (SBB) treatment. Results The AF within the different rat intestinal segments was easily detected in both FITC- and TRITC-channel spectrum, which was not affected by serum blocking. After 0.3% SBB treatment for 10 minutes, a significant reduction of AF was observed in the rat intestinal tissue. Moreover, the SBB treatment did not affect the expression of CD45 in the rat intestine detected by IF staining, but reduced the AF remarkedly. Conclusion The AF in rat intestinal frozen section can be effectively reduced with 0.3% SBB treatment for 10 minutes.

导出引用

李勇周立张阳刘震姚念申凤鸽王志勇. 苏丹黑B降低大鼠肠道自发荧光及其在免疫荧光染色中的应用[J]. 解剖学报. 2020, 51(3): 450-455 https://doi.org/10.16098/j.issn.0529-1356.2020.03.024

LI Yong ZHOU Li ZHANG Yang LIU Zhen YAO Nian SHEN Feng-ge WANG Zhi-yong. Sudan black B reducing the intestinal autofluorescence of rats and its application in immunofluorescence[J]. Acta Anatomica Sinica. 2020, 51(3): 450-455 https://doi.org/10.16098/j.issn.0529-1356.2020.03.024

中图分类号： R338.2

参考文献

［1］ Billinton N, Knight AW. Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence ［J］. Anal Biochem, 2001, 291(2): 175-197.

［2］ Vida C, De Toda IM, Cruces J, et al. Role of macrophages in age-related oxidative stress and lipofuscin accumulation in mice ［J］. Redox Biol, 2017, 12(1): 423-437.

［3］ Barnes D, Aggarwal S, Thomsen S, et al. A characterization of the fluorescent properties of circulating human eosinophils ［J］. Photochem Photobiol, 1993, 58(2): 297-303.

［4］ Lei L, Tzekov R, Tang S, et al. Accumulation and autofluorescence of phagocytized rod outer segment material in macrophages and microglial cells ［J］. Mol Vis, 2012, 18:103-113.

［5］ Tang XT, Ibanez F, Tamborindeguy C. Quenching autofluorescence in the alimentary canal tissues of Bactericera cockerelli (Hemiptera: Triozidae) for immunofluorescence labeling ［J］. Insect Sci, 2019, 1(1): 1-12.

［6］ Baschong W, Suetterlin R, Laeng RH. Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM) ［J］. J Histochem Cytochem, 2001, 49(12): 1565-1572.

［7］ Qin W, Luo M, Wang K, et al. A combined treatment with erythrocyte lysis solution and Sudan Black B reduces tissue autofluorescence in double-labeling immunofluorescence ［J］. Microscopy (Oxf), 2018, 67(6): 345-355.

［8］ Liu XN, Zhu XD, Zhao J, et al. The effect of GnRH-A on the function of gastrin cells in digestive tract of rats ［J］. Acta Anatomica Sinica, 2004,35(2):190-193.（in Chinese）

刘晓宁,朱晓东,赵洁,等.胃腔内促性腺激素释放激素类似物对大鼠消化道胃泌素细胞功能的影响［J］.解剖学报,2004,35(2):190-193.

［9］ Mcghee JR, Fujihashi K. Inside the mucosal immune system ［J］. PLoS Biol, 2012, 10(9): e1001397.

［10］ Wizenty J, Ashraf MI, Rohwer N, et al. Autofluorescence: a potential pitfall in immunofluorescence-based inflammation grading ［J］. J Immunol Methods, 2018, 456(1): 28-37.

［11］ Jenvey CJ, Stabel JR. Autofluorescence and nonspecific immunofluorescent labeling in frozen bovine intestinal tissue sections: solutions for multicolor immunofluorescence experiments ［J］. J Histochem Cytochem, 2017, 65(9): 531-541.

［12］ Evangelou K, Gorgoulis VG. Sudan black B, the specific histochemical stain for lipofuscin: a novel method to detect senescent cells ［J］. Methods Mol Biol, 2017, 1534:111-119.

［13］ Oliveira VC, Carrara RC, Simoes DL, et al. Sudan black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections ［J］. Histol Histopathol, 2010, 25(8): 1017-1024.

［14］ Altin JG, Sloan EK. The role of CD45 and CD45-associated molecules in T cell activation ［J］. Immunol Cell Biol, 1997, 75(5): 430-445.