Methanol, 95% ethanol, 4% PFA, acetone and glutaraldehyde are routinely used as fixatives in biomedical experiments. In many studies, cells or tissues need to be fixed for subsequent microscopic observation, probe hybridization, or immunochemical staining. Before the relevant experiments are performed, the corresponding fixatives should be selected according to the specific cells, tissues and experimental purposes. FPs are applied as reporter genes and are widely used in gene transfection and expression studies and for in vivo fluorescence imaging of transgenic animals to observe the movement and localization of target proteins in cells. In addition, transgenic fluorescent animal models provide powerful tools for molecular imaging studies and studies on tumor biology, immune-tumor cell interaction, tumor angiogenesis, and stem cell differentiation and localization. Although FPs are widely applied in biomedical research, few studies have reported changes in the fluorescence intensity of FPs during cell or tissue fixation. Only sporadic information in relevant reports is available to researchers as guidance. Unfortunately, the descriptions of cell or tissue fixation methods in most studies are very simple, making it difficult for other experimenters to accurately determine the methods that were used. Therefore, the effects of some routinely used fixatives on the fluorescence intensity of FPs need to be elucidated.
Fixatives commonly applied in general molecular biology experiments are divided into two categories: organic solvents and crosslinkers18. These fixatives are used for research and diagnostics and act through different stabilization mechanisms. The main organic solvent fixatives are methanol, ethanol and acetone. Methanol is a strong dehydrating agent that can immobilize cells in a certain configuration and increase the surface area of cell structures19. Ethanol is a small-molecule nonpolar permeabilizing substance that can penetrate cells quickly, fix the fine structures of cells, dehydrate tissues/cells, and denature proteins via precipitation to fix cells20. Acetone is a fixative with permeabilization, strong dehydration, and protein precipitation abilities19. Paraformaldehyde and glutaraldehyde are routinely used crosslinking agents. The aldehyde group in the paraformaldehyde molecule binds with the amino groups of proteins to form carboxymethyl groups, thus forming intermolecular crosslinks that affect protein configurations and fix the proteins21. Glutaraldehyde is a bifunctional crosslinking agent22 that has two reactive aldehyde groups that can undergo acylation with amino groups, mercapto groups and other groups, enabling it to crosslink with proteins. The changes in the molecular structure and conformation of cells induced by fixation may change the fluorescence intensity and protein interactions of FPs. Therefore, selection of an appropriate fixative is crucial for accurate detection of the protein of interest and its biological function.
Previous studies have reported that the GFP signal in GFP-expressing cells can be successfully maintained using PFA23–24. Our study showed that FP fluorescence decreased after fixation with 4% PFA at room temperature and that CFP and exhibited the most significant fluorescence loss, while strong signals were preserved for GFP and RFP. According to some studies, FP signals decrease or disappear after fixation with 4% PFA; these findings may be related to deviation from a neutral pH during the preparation of 4% PFA. The pH value of 4% PFA in this experiment was close to neutral, so interference of the pH value with the experimental results was excluded. In another study with 4°C PFA, the GFP signal was barely detectable, indicating that crosslinking of proteins was too slow to retain GFP; in contrast, more confined and intense GFP signals were obtained in sections postfixed with 25°C PFA25. Interestingly, another fixative called glyoxal has recently been proposed to be a relevant alternative to PFA, but whether glyoxal works as a postfixative to effectively preserve FP signals remains to be investigated. Few studies have reported the effect of glutaraldehyde on FP signals. In this study, glutaraldehyde also decreased FP fluorescence; approximately 1/3 of the fluorescence was preserved.
Previous studies have shown that acetone can preserve GFP signals as well. The results of our study showed that acetone weakened the FP signals, among which the GFP and RFP signals decreased most obviously. However, the CFP and YFP signals remained strong. The different mechanisms of acetone and paraformaldehyde fixation in cells provide choices for experimental studies using different FPs as gene probes.
Some studies have reported that methanol can also be applied to maintain GFP fluorescence26, but our study showed that methanol may lead to weakening or even disappearance of GFP fluorescence10. In fact, the fluorescence signals of all four FPs decreased significantly after fixation with methanol, indicating that methanol could not effectively preserve the FP signals. Therefore, methanol is not recommended for fixation of cells expressing FPs.
Ethanol-mediated cell fixation has been found to cause GFP fluorescence to disappear quickly5. In agreement with this finding, our study revealed that the fluorescence intensity of the four FPs was weakened rapidly after fixation with 95% ethanol. Among the signals, the RFP signal was lost most obviously, while some of the fluorescence intensity of CFP was preserved. Some studies have reported that methanol and ethanol alone or mixed with acetone and formaldehyde can be used as fixatives. However, unless otherwise required for specific experiments, for the types of FPs applied in our study, acetone is recommended for fixation to maintain CFP and YFP fluorescence, while 4% PFA is recommended for fixation to preserve GFP and YFP fluorescence.
Another question is whether increases or decreases in FP fluorescence during fixation will affect the results of related experiments based on the fluorescence intensity of FPs. To answer this question, we selected two interacting proteins, ADORA2a and DRD2, for co-expression with YFP and CFP, respectively. We co-transfected them into HEK293T cells with lentiviruses and then detected the FRET efficiency. The optimum distance of FRET detection is 1–10 nm; when the spatial displacement of FP molecules is too large, it affects the detection efficiency of FRET, especially for cells with strong molecular motion of FPs. In such cases, it is necessary to fix the cells in order to retain the interactions between protein molecules in a particular cell state and to perform the FRET experiment smoothly.
In our study, the fixatives had significant negative impacts on FRET efficiency (all P < 0.01). The FRET efficiency after 95% ethanol fixation was higher than that before fixation, while the efficiencies after methanol, 4% PFA, acetone and glutaraldehyde fixation were all lower than those before fixation. The FRET efficiency decreased most obviously after fixation with 4% PFA. Previous studies have shown no significant differences in FRET efficiency between unfixed living cells and living cells fixed with 4% PFA27. Chu Y W et al.24 showed that FRET efficiency decreased after PFA fixation, consistent with the results of this study. The effects may be related to the mechanisms of the fixatives; the specific mechanisms need further study.
This experiment also used 4% PFA-fixed brain tissue sections of transgenic mice labeled with EGFP and Ai4 transgenic mice labeled with tdTomato to explore the effect of fixatives on FP signals. The results showed that the fluorescence signals of EGFP and tdTomato in transgenic fluorescent mouse brain tissues decreased after fixation with 4% PFA.
According to the results of our study, the effects of fixatives on FP signals in cultured cells and transgenic fluorescent mouse tissues are unfavorable to some extent. Our findings also confirmed that these effects can cause bias in FRET efficiency. Whether the effects also cause errors in the results of other experiments based on the fluorescence intensities of FPs, such as flow cytometry or immunohistochemistry, remains to be examined.
Taken together, our findings reveal that fixatives indeed have certain unfavorable effects on FP signals in cells and tissues that lead to decreases in or even loss of their biological functions. Therefore, fixatives should be applied carefully in FP-related experiments to avoid bias in the experimental results.