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Improved quantification of immune-gold labeling and its use to compare the distribution of cellular factors among sub-chloroplast compartments

文献类型： 外文期刊

作者： Lv, Ming-fang 1 ; Xie, Li 2 ; Li, Yun-qin 2 ; Zhang, Xiao-min 2 ; Li, Mei 2 ; Liao, Zhen-feng 3 ; Zhang, Zhong-kai 4 ; Ho 1 ;

作者机构： 1.Zhejiang Acad Agr Sci, Inst Virol & Biotechnol, Hangzhou 310021, Peoples R China

2.Zhejiang Univ, Anal Ctr Agrobiol & Environm Sci, Hangzhou 310058, Peoples R China

3.Zhejiang Acad Agr Sci, Publ Lab, Hangzhou 310021, Peoples R China

4.Yunnan Acad Agr Sci, Inst Biotechnol & Genet Resources, Kunming 650223, Yunnan, Peoples R China

关键词： Immuno-gold labeling; Quantification; Sub-chloroplast compartment; Image processing; Background subtraction; Distribution frequency

期刊名称：MICRON （ 影响因子：2.251； 五年影响因子：1.969 ）

ISSN： 0968-4328

年卷期： 2021 年 145 卷

页码：

收录情况： SCI

摘要： Quantification of immuno-gold labeling can provide valuable information on the quantity and localization of a target within a region of interest (ROI). Background subtraction usually requires preparation of material with a deliberately reduced amount of target component often by gene knockout/knockdown. This paper reports a modified method without the need for gene knockout/knockdown, by using a region outside the ROI as a background and non-immune serum to verify the reliability of the data. An optimized parameter for use in image processing was also developed to improve semi-automatic segmentation of gold particles, by using the standard deviation of pixel intensity together with default parameters (size and intensity) to improve specificity. The modified methods were used to quantify the gold labeling of various components within chloroplasts and their 3 sub-organelle compartments (thylakoid, stroma and starch). Rubisco, actin, myosin, beta-tubulin, Endoplasmic reticulum-retention signal HDEL, Sterol methyltransferase 1, and double stranded RNA were all effectively and consistently quantified at the level of the different sub-chloroplast compartments. The approach should be applicable more widely for high resolution labelling of samples in which a background requiring gene knockout/ knockdown is not a realistic option.

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