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Spectral Characteristics of Autofluorescence in Renal Tissue and Methods for Reducing Fluorescence Background in Confocal Laser Scanning Microscopy

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Abstract

Significant autofluorescence (AF) of renal tissue is one of the major causes restricting the use of immunofluorescent staining. This study aimed at controlling renal tissue AF and testing an effective method for optimizing specific signals. In the present study, we observed emergence of strong AF in all renal cells under different fluorescent channels. Significant concentration-dependent reduction in AF of kidney tissue was observed with the use of sodium borohydride (NaBH4) and Sudan black B (SBB) alone (p < 0.05). Under maximum effective concentration, semi-quantitative analysis revealed that inhibitory effect of SBB on AF was superior to that of NaBH4 (P < 0.01). When the two chemicals were combined, we observed that background can be reduced, and specific staining can be optimized at optimum concentration. Intensity of renal tissue was examined by confocal λ scanning, which showed that peaks were located at the range of approximately 480 − 590 nm and similar to those of flavin and lipofuscin. These results indicated that combined use of NaBH4 and SBB, when targeted at different sources of AF in renal tissue, is the most effective means of reducing background and preserving specificity of fluorescent labels. In addition, this method does not interfere with various steps of immunofluorescence experiments.

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Acknowledgements

The present study was supported by National Natural Science Foundation of China (No. 81560175 and No. 81260159), the High Level Talent Research Project of Shihezi University (grant no. RCSX201705), and Xinjiang Autonomous Region Graduate Research and Innovation Project (grant no. XJGRI2017033).

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  1. Yang Zhang and Yang Wang contributed equally to this work.

Authors and Affiliations

  1. Department of Physiology, Shihezi University Medical College, 192 Beier Road, Shihezi, Xinjiang, 832002, People’s Republic of China

    Yang Zhang, Yang Wang, Ke-Tao Ma, Wei Ji, Zi-Wei Han, Jun-Qiang Si & Li Li

  2. The Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Medical College of Shihezi University, Shihezi, 832002, China

    Yang Wang, Wei-Wei Cao, Ke-Tao Ma, Jun-Qiang Si & Li Li

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  1. Yang Zhang
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Correspondence to Jun-Qiang Si or Li Li.

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Zhang, Y., Wang, Y., Cao, WW. et al. Spectral Characteristics of Autofluorescence in Renal Tissue and Methods for Reducing Fluorescence Background in Confocal Laser Scanning Microscopy. J Fluoresc 28, 561–572 (2018). https://doi.org/10.1007/s10895-018-2217-4

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