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bioRxiv · 10.1101/2025.09.22.677869

Preservation of soluble enhanced green fluorescent protein (EGFP) within fixed NIH 3T3 fibroblasts

Elliott, J. T.·Mohiuddin, M.·Tona, A.
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Abstract

Quantitative imaging of cytoplasmic green fluorescence protein (GFP) in fixed cells can be unreliable if the fixing process does not preserve the total fluorescence intensity level or the spatial location relative to the living cells. In this study, we examine the effect of fixatives (formaldehyde, disuccinimidyl glutarate (DSG), dithiobis(succinimidyl) propionate (DSP) and m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS)), fixative buffers and the cross-linking times on the fluorescent intensity of soluble enhanced GFP (EGFP) expressed within NIH 3T3 cells. The total fluorescence intensity within individual cells during the fixation process was measured in an automated fluorescence microscope. Our results show that choice of fixative, the fixing solution and the cross-linking time were important for minimizing EGFP losses during fixing. The optimal fixation condition for these cells was identified to be the MBS cross-linker in a microtubule stabilizing buffer. After an 8 h fixation, greater than 90 % of the initial GFP fluorescence within cells was preserved. This was 3-fold higher than the GFP fluorescence remaining when the cells were fixed with 1 % paraformaldehyde in PBS. MBS treated cells could be permeabilized with 0.05 % Triton X-100 with little additional loss in fluorescence intensity. The MBS fixative could also preserve soluble fluorescent proteins in suspension cells, suggesting the fixation results are not cell line-specific. Direct imaging of the fixation process on fluorescent reporter cells provided insight into the effect of chemical fixatives on biological cells and can facilitate identification of fixation protocols that are fit-for-purpose for quantitative measurements.

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Elliott, J. T., Mohiuddin, M., Tona, A.. 2025-09-23. Preservation of soluble enhanced green fluorescent protein (EGFP) within fixed NIH 3T3 fibroblasts. https://doi.org/10.1101/2025.09.22.677869

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