Unfavorable Effects of Fixatives on the Fluorescence Intensity and Biological Functions of Fluorescent Proteins in HEK293T Cells and Transgenic Mice

Fluorescent proteins (FPs) are commonly used probes for coding genes that enable specific protein or whole-cell labeling. Their fluorescence intensity is used for molecular quantitation and intermolecular interaction analysis. Since FPs are usually small soluble proteins, they easily cross the membranes if cell integrity is disrupted, resulting in FP signal attenuation/loss. Specimen prefixation to preserve FP localization within cells/tissues is therefore useful. However, specific fixatives can weaken or eliminate FP signals. We studied the effects of five common fixatives on FP fluorescence intensity and biological functions to determine their suitability for FP signal and FRET efficiency preservation in cells and tissues. FP (GFP, YFP, CFP and RFP)-expressing HEK293T cells with methanol, 95% ethanol, 4% PFA, acetone and glutaraldehyde, and brain tissue sections of EGFP- and tdTomato-labeled transgenic fluorescent mice was fixed with 4% PFA. The FP signals in HEK293T cells and brain tissue from transgenic fluorescent mice were weakened or even eliminated after fixation with these fixatives. The fixatives affected FP biological function, and the FP FRET efficiency significantly differed between prefixation and postfixation (all p<0.01). Thus, fixatives impair FP fluorescence to some extent, leading to attenuation/loss of signals or even biological functions. Fixatives should be applied carefully in FP-related experiments to avoid bias.