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Determination of the lowest concentrations of aldehyde fixatives for completely fixing various cellular structures by real-time imaging and quantification

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Abstract

The effectiveness of fixatives for fixing biological specimens has long been widely investigated. However, the lowest concentrations of fixatives needed to completely fix whole cells or various cellular structures remain unclear. Using real-time imaging and quantification, we determined the lowest concentrations of glutaraldehyde (0.001–0.005, ~0.005, 0.01–005, 0.01–005, and 0.01–0.1 %) and formaldehyde/paraformaldehyde (0.01–0.05, ~0.05, 0.5–1, 1–1.5, and 0.5–1 %) required to completely fix focal adhesions, cell-surface particles, stress fibers, the cell cortex, and the inner structures of human umbilical vein endothelial cells within 20 min. With prolonged fixation times (>20 min), the concentration of fixative required to completely fix these structures will shift to even lower values. These data may help us understand and optimize fixation protocols and understand the potential effects of the small quantities of endogenously generated aldehydes in human cells. We also determined the lowest concentration of glutaraldehyde (0.5 %) and formaldehyde/paraformaldehyde (2 %) required to induce cell blebbing. We found that the average number and size of the fixation-induced blebs per cell were dependent on both fixative concentration and cell spread area, but were independent of temperature. These data provide important information for understanding cell blebbing, and may help optimize the vesiculation-based technique used to isolate plasma membrane by suggesting ways of controlling the number or size of fixation-induced cell blebs.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (30900340), the Natural Science Foundation of Jiangxi Province (2010GZN0138), the Scientific Research Foundation for Returned Overseas Chinese Scholar of State Education Ministry, and the Scientific Research Fund of Jiangxi Provincial Education Department (GJJ10305). We thank Sherry S. Chen for promoting the manuscript.

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Authors and Affiliations

  1. Institute for Advanced Study, Nanchang University, Nanchang, 330031, China

    Fangfa Zeng, Wen Yang, Jie Huang, Yuan Chen & Yong Chen

  2. Department of Biotechnology, Nanchang University, Nanchang, 330031, China

    Fangfa Zeng, Jie Huang & Yong Chen

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  1. Fangfa Zeng
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  2. Wen Yang
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  3. Jie Huang
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  4. Yuan Chen
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Correspondence to Yong Chen.

Electronic supplementary material

Below is the link to the electronic supplementary material.

418_2012_1058_MOESM1_ESM.avi

Video S1 Dynamic imaging of cold-induced cell contraction of unfixed HUVECs. Cells were imaged for 20 min. (AVI 5300 kb)

418_2012_1058_MOESM2_ESM.avi

Video S2 Dynamic imaging of cold-induced cell contraction of cells fixed with 0.001% glutaraldehyde. Cells were imaged for 20 min. (AVI 4382 kb)

418_2012_1058_MOESM3_ESM.avi

Video S3 Dynamic imaging of cold-induced cell contraction of cells fixed with 0.005% glutaraldehyde. Cells were imaged for 20 min. (AVI 3397 kb)

418_2012_1058_MOESM4_ESM.avi

Video S4 Dynamic imaging of swelling-induced cell contraction of unfixed HUVECs. Cells were imaged for 10 min. (AVI 2658 kb)

418_2012_1058_MOESM5_ESM.avi

Video S5 Dynamic imaging of swelling-induced cell contraction of cells prefixed with 0.001% glutaraldehyde. Cells were imaged for 10 min. (AVI 5081 kb)

418_2012_1058_MOESM6_ESM.avi

Video S6 Dynamic imaging of swelling-induced cell contraction of cells prefixed with 0.1% formaldehyde. Cells were imaged for 10 min. (AVI 4022 kb)

418_2012_1058_MOESM7_ESM.avi

Video S7 Dynamic imaging of cell-surface particle movement completely stopping in cells fixed with 0.005% glutaraldehyde. Cells were imaged for 20 min. (AVI 4008 kb)

418_2012_1058_MOESM8_ESM.avi

Video S8 Dynamic imaging of cell-surface particle movement completely stopping in cells fixed with 0.05% formaldehyde. Cells were imaged for 20 min. (AVI 3291 kb)

418_2012_1058_MOESM9_ESM.avi

Video S9 Dynamic imaging of fixation-induced blebbing on cells fixed with 4% glutaraldehyde. Cells were imaged for 20 min. (AVI 4547 kb)

418_2012_1058_MOESM10_ESM.avi

Video S10 Dynamic imaging of fixation-induced blebbing on cells fixed with 4% formaldehyde. Cells were imaged for 20 min. (AVI 5266 kb)

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Zeng, F., Yang, W., Huang, J. et al. Determination of the lowest concentrations of aldehyde fixatives for completely fixing various cellular structures by real-time imaging and quantification. Histochem Cell Biol 139, 735–749 (2013). https://doi.org/10.1007/s00418-012-1058-5

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