Abstract

Resource16 November 2017Open Access Source DataTransparent process Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy Katharina N Richter Katharina N Richter Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Göttingen, Germany Search for more papers by this author Natalia H Revelo Natalia H Revelo orcid.org/0000-0003-2492-1317 Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany Search for more papers by this author Katharina J Seitz Katharina J Seitz Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany International Max Planck Research School Molecular Biology, Göttingen, Germany Search for more papers by this author Martin S Helm Martin S Helm Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany International Max Planck Research School Molecular Biology, Göttingen, Germany Search for more papers by this author Deblina Sarkar Deblina Sarkar MIT Media Lab Search for more papers by this author Rebecca S Saleeb Rebecca S Saleeb Edinburgh Super-Resolution Imaging Consortium, Institute of Biological Chemistry, Biophysics, and Bioengineering, Heriot-Watt University, Edinburgh, UK Search for more papers by this author Elisa D'Este Elisa D'Este Department of NanoBiophotonics, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Jessica Eberle Jessica Eberle Department of Neural Systems, Max-Planck-Institute for Brain Research, Frankfurt am Main, Germany Search for more papers by this author Eva Wagner Eva Wagner Heart Research Center Göttingen, Department of Cardiology & Pulmonology, University Medical Center Göttingen, Göttingen, Germany German Center for Cardiovascular Research (DZHK) Site Göttingen Search for more papers by this author Christian Vogl Christian Vogl orcid.org/0000-0003-4432-2733 Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Max-Planck-Institute for Experimental Medicine, Auditory Neuroscience Group, Göttingen, Germany Search for more papers by this author Diana F Lazaro Diana F Lazaro Department of Experimental Neurodegeneration, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany Max-Planck-Institute for Experimental Medicine, Göttingen, Germany Search for more papers by this author Frank Richter Frank Richter International Max Planck Research School Molecular Biology, Göttingen, Germany Department of Cellular Biochemistry, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Javier Coy-Vergara Javier Coy-Vergara Department of Molecular Biology, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Giovanna Coceano Giovanna Coceano Department of Applied Physics and Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden Search for more papers by this author Edward S Boyden Edward S Boyden Departments of Brain and Cognitive Science and Biological Engineering, MIT Media Lab and McGovern Institute, Cambridge, MA, USA Search for more papers by this author Rory R Duncan Rory R Duncan Edinburgh Super-Resolution Imaging Consortium, Institute of Biological Chemistry, Biophysics, and Bioengineering, Heriot-Watt University, Edinburgh, UK Search for more papers by this author Stefan W Hell Stefan W Hell Department of NanoBiophotonics, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Marcel A Lauterbach Marcel A Lauterbach Department of Neural Systems, Max-Planck-Institute for Brain Research, Frankfurt am Main, Germany Search for more papers by this author Stephan E Lehnart Stephan E Lehnart Heart Research Center Göttingen, Department of Cardiology & Pulmonology, University Medical Center Göttingen, Göttingen, Germany German Center for Cardiovascular Research (DZHK) Site Göttingen Search for more papers by this author Tobias Moser Tobias Moser orcid.org/0000-0001-7145-0533 Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Max-Planck-Institute for Experimental Medicine, Auditory Neuroscience Group, Göttingen, Germany Search for more papers by this author Tiago F Outeiro Tiago F Outeiro orcid.org/0000-0003-1679-1727 Department of Experimental Neurodegeneration, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany Max-Planck-Institute for Experimental Medicine, Göttingen, Germany Search for more papers by this author Peter Rehling Peter Rehling Department of Cellular Biochemistry, University Medical Center Göttingen, Göttingen, Germany Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Blanche Schwappach Blanche Schwappach Department of Molecular Biology, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Ilaria Testa Ilaria Testa Department of Applied Physics and Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden Search for more papers by this author Bolek Zapiec Bolek Zapiec Max Planck Research Unit for Neurogenetics, Frankfurt am Main, Germany Search for more papers by this author Silvio O Rizzoli Corresponding Author Silvio O Rizzoli [email protected] orcid.org/0000-0002-1667-7839 Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Göttingen, Germany Search for more papers by this author Katharina N Richter Katharina N Richter Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Göttingen, Germany Search for more papers by this author Natalia H Revelo Natalia H Revelo orcid.org/0000-0003-2492-1317 Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany Search for more papers by this author Katharina J Seitz Katharina J Seitz Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany International Max Planck Research School Molecular Biology, Göttingen, Germany Search for more papers by this author Martin S Helm Martin S Helm Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany International Max Planck Research School Molecular Biology, Göttingen, Germany Search for more papers by this author Deblina Sarkar Deblina Sarkar MIT Media Lab Search for more papers by this author Rebecca S Saleeb Rebecca S Saleeb Edinburgh Super-Resolution Imaging Consortium, Institute of Biological Chemistry, Biophysics, and Bioengineering, Heriot-Watt University, Edinburgh, UK Search for more papers by this author Elisa D'Este Elisa D'Este Department of NanoBiophotonics, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Jessica Eberle Jessica Eberle Department of Neural Systems, Max-Planck-Institute for Brain Research, Frankfurt am Main, Germany Search for more papers by this author Eva Wagner Eva Wagner Heart Research Center Göttingen, Department of Cardiology & Pulmonology, University Medical Center Göttingen, Göttingen, Germany German Center for Cardiovascular Research (DZHK) Site Göttingen Search for more papers by this author Christian Vogl Christian Vogl orcid.org/0000-0003-4432-2733 Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Max-Planck-Institute for Experimental Medicine, Auditory Neuroscience Group, Göttingen, Germany Search for more papers by this author Diana F Lazaro Diana F Lazaro Department of Experimental Neurodegeneration, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany Max-Planck-Institute for Experimental Medicine, Göttingen, Germany Search for more papers by this author Frank Richter Frank Richter International Max Planck Research School Molecular Biology, Göttingen, Germany Department of Cellular Biochemistry, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Javier Coy-Vergara Javier Coy-Vergara Department of Molecular Biology, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Giovanna Coceano Giovanna Coceano Department of Applied Physics and Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden Search for more papers by this author Edward S Boyden Edward S Boyden Departments of Brain and Cognitive Science and Biological Engineering, MIT Media Lab and McGovern Institute, Cambridge, MA, USA Search for more papers by this author Rory R Duncan Rory R Duncan Edinburgh Super-Resolution Imaging Consortium, Institute of Biological Chemistry, Biophysics, and Bioengineering, Heriot-Watt University, Edinburgh, UK Search for more papers by this author Stefan W Hell Stefan W Hell Department of NanoBiophotonics, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Marcel A Lauterbach Marcel A Lauterbach Department of Neural Systems, Max-Planck-Institute for Brain Research, Frankfurt am Main, Germany Search for more papers by this author Stephan E Lehnart Stephan E Lehnart Heart Research Center Göttingen, Department of Cardiology & Pulmonology, University Medical Center Göttingen, Göttingen, Germany German Center for Cardiovascular Research (DZHK) Site Göttingen Search for more papers by this author Tobias Moser Tobias Moser orcid.org/0000-0001-7145-0533 Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Max-Planck-Institute for Experimental Medicine, Auditory Neuroscience Group, Göttingen, Germany Search for more papers by this author Tiago F Outeiro Tiago F Outeiro orcid.org/0000-0003-1679-1727 Department of Experimental Neurodegeneration, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany Max-Planck-Institute for Experimental Medicine, Göttingen, Germany Search for more papers by this author Peter Rehling Peter Rehling Department of Cellular Biochemistry, University Medical Center Göttingen, Göttingen, Germany Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Blanche Schwappach Blanche Schwappach Department of Molecular Biology, University Medical Center Göttingen, Göttingen, Germany Search for more papers by this author Ilaria Testa Ilaria Testa Department of Applied Physics and Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden Search for more papers by this author Bolek Zapiec Bolek Zapiec Max Planck Research Unit for Neurogenetics, Frankfurt am Main, Germany Search for more papers by this author Silvio O Rizzoli Corresponding Author Silvio O Rizzoli [email protected] orcid.org/0000-0002-1667-7839 Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Göttingen, Germany Search for more papers by this author Author Information Katharina N Richter1,2,‡, Natalia H Revelo1,20,‡, Katharina J Seitz1,3, Martin S Helm1,3, Deblina Sarkar4, Rebecca S Saleeb5, Elisa D'Este6, Jessica Eberle7, Eva Wagner8,9, Christian Vogl10,11, Diana F Lazaro12,13, Frank Richter3,14, Javier Coy-Vergara15, Giovanna Coceano16, Edward S Boyden17, Rory R Duncan5, Stefan W Hell6, Marcel A Lauterbach7, Stephan E Lehnart8,9, Tobias Moser10,11, Tiago F Outeiro12,13, Peter Rehling14,18, Blanche Schwappach15, Ilaria Testa16, Bolek Zapiec19 and Silvio O Rizzoli *,1,2 1Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany 2Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Göttingen, Germany 3International Max Planck Research School Molecular Biology, Göttingen, Germany 4MIT Media Lab 5Edinburgh Super-Resolution Imaging Consortium, Institute of Biological Chemistry, Biophysics, and Bioengineering, Heriot-Watt University, Edinburgh, UK 6Department of NanoBiophotonics, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany 7Department of Neural Systems, Max-Planck-Institute for Brain Research, Frankfurt am Main, Germany 8Heart Research Center Göttingen, Department of Cardiology & Pulmonology, University Medical Center Göttingen, Göttingen, Germany 9German Center for Cardiovascular Research (DZHK) Site Göttingen 10Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany 11Max-Planck-Institute for Experimental Medicine, Auditory Neuroscience Group, Göttingen, Germany 12Department of Experimental Neurodegeneration, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany 13Max-Planck-Institute for Experimental Medicine, Göttingen, Germany 14Department of Cellular Biochemistry, University Medical Center Göttingen, Göttingen, Germany 15Department of Molecular Biology, University Medical Center Göttingen, Göttingen, Germany 16Department of Applied Physics and Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden 17Departments of Brain and Cognitive Science and Biological Engineering, MIT Media Lab and McGovern Institute, Cambridge, MA, USA 18Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany 19Max Planck Research Unit for Neurogenetics, Frankfurt am Main, Germany 20Present address: Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands ‡These authors contributed equally to this work *Corresponding author. Tel: +49 551 395911; E-mail: [email protected] The EMBO Journal (2018)37:139-159https://doi.org/10.15252/embj.201695709 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA. Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining. Synopsis Enhanced fixation speed, decreased toxicity and better preservation of cytosolic nanostructures and membrane-bound antigens make the small dialdehyde glyoxal a promising alternative to the commonly used paraformaldehyde (PFA), in particular for super-resolution microscopy. Glyoxal penetrates cells faster than conventional solutions of PFA. Glyoxal fixes and retains more cellular proteins and RNA than PFA. Immunostaining of glyoxal-fixed samples reveals higher amounts of target proteins, and typically results in brighter microscopy images. Glyoxal fixation was validated against multiple PFA-based protocols in several laboratories. Introduction The 4% paraformaldehyde (PFA) solution has been a standard fixative for immunostaining and fluorescence microscopy, for several decades. Nevertheless, the literature contains numerous reports that PFA causes morphological changes, loss of epitopes, or mislocalization of target proteins and that it fixes the samples slowly and incompletely (see, e.g., Melan, 1994; Tanaka et al, 2010; Schnell et al, 2012). Many other fixatives have been introduced to alleviate these problems. Among them, glutaraldehyde is probably the most commonly used, since it fixes the samples faster and more completely than PFA (Smith & Reese, 1980). Mixtures of PFA and glutaraldehyde result in accurate fixation and reduce the lateral mobility of molecules (Tanaka et al, 2010), presumably by increasing the level of protein cross-linking. However, this fixative mixture also reduces the efficiency of immunostainings, by blocking the antibody access to epitopes, or by causing particular epitopes to unfold (Farr & Nakane, 1981). Alcohol-based fixation, such as treatments with ice-cold methanol (Tanaka et al, 2010), results in stable fixation for a sub-population of cellular structures (such as microtubules), but leads to poor morphology preservation and to a loss of membranes and cytosolic proteins. Overall, the improvements in fixation induced by glutaraldehyde or methanol do not compensate for their shortcomings, thus in most cases leaving PFA as the current fixative of choice. A superior alternative to PFA is needed, especially since artifacts that were negligible in conventional microscopy are now rendered visible by the recent progress in super-resolution microscopy (nanoscopy; Eggeling et al, 2015). To find a fixative that maintains high-quality immunostainings while alleviating PFA problems, we have tested several compounds. We searched for commercially available molecules, which could be readily used by the imaging community. These included different combinations of PFA and glutaraldehyde, picric acid (Hopwood, 1985), and di-imido-esters (Woodruff & Rasmussen, 1979), which, however, were not better than PFA in immunostaining experiments. We have also investigated different aldehydes. We avoided highly toxic compounds such as acrolein, which would not be easy to use in biology laboratories, and we also avoided large aldehydes (more than 4–5 carbon atoms), whose fixative properties are expected to mimic those of glutaraldehyde. The small dialdehyde glyoxal fits these two criteria, since it has a low toxicity (as already noted in the 1940s, Wicks & Suntzeff, 1943) and contains only two carbon atoms. Glyoxal is used, at low concentrations, in glycation and metabolism studies (Boucher et al, 2015), which ensures that it is commercially available. It can be used as a fixative and has even been once described, in 1963, to provide better morphology preservation to formaldehyde (Sabatini et al, 1963). It is almost unknown in fluorescence experiments. We were able to find one publication, from 1975 (Swaab et al, 1975), in which glyoxal was used in immunofluorescence on brain samples, albeit followed by sample freezing, and by procedures that are not compatible with modern, high-quality microscopy. We could also find a few publications on histological stains using glyoxal (e.g., Umlas & Tulecke, 2004; Paavilainen et al, 2010), which further encouraged us to test this compound. We tested glyoxal thoroughly, in preparations ranging from cell-free cytosol to tissues, and by methods spanning from SDS–PAGE to electron microscopy and super-resolution fluorescence microscopy. We found that glyoxal penetrated cells far more rapidly than PFA and cross-linked proteins and nucleic acids more strongly, leading to a more accurate preservation of cellular morphology. Despite the stronger fixation, glyoxal did not cause a reduction of antibody binding to the samples. On the contrary, the resulting images were typically brighter than those obtained after PFA fixation. The initial optimization work was performed in one laboratory (Rizzoli, University Medical Center Göttingen, Germany), and the results were independently tested in 11 additional laboratories/teams: Boyden (MIT Media Lab and McGovern Institute, Massachusetts, USA), Duncan (Heriot-Watt University, Edinburgh, UK), Hell (Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany), Lauterbach (Max-Planck-Institute for Brain Research, Frankfurt am Main, Germany), Lehnart (University Medical Center Göttingen, Germany), Moser (University Medical Center Göttingen, Germany), Outeiro (University Medical Center Göttingen, Germany), Rehling (University Medical Center Göttingen, Germany), Schwappach (University Medical Center Göttingen, Germany), Testa (KTH Royal Institute of Technology, Stockholm, Sweden), and Zapiec (Max Planck Research Unit for Neurogenetics, Frankfurt am Main, Germany). We conclude that the immunostainings performed after glyoxal fixation were superior for the majority of the samples and targets, with only a minority (~10%) of the targets being less well preserved and/or revealed. Results Glyoxal preserves the cellular morphology more accurately than PFA and fixes proteins and RNAs more strongly To determine the optimal conditions of glyoxal fixation, we tested its action at different pH values (Appendix Table S1). We found that glyoxal requires an acidic pH, roughly between 4 and 5, despite one previous study that suggests that it may also fix samples at a neutral pH (Sabatini et al, 1963). In addition, we found that the morphology of the samples was much improved upon addition of a low-to-medium concentration of alcohol (ethanol, 10–20%), which may act as an accelerator in the fixation reactions. Removing the ethanol, or adjusting the pH above or below the 4–5 range, resulted in poor sample morphology (Appendix Table S1). pH values of 4 or 5 provided similar results for most of our experiments (results obtained at pH 5 are shown in all figures, unless noted otherwise) and provided better morphology preservation for cultured neurons than PFA. We tested PFA at various pH values (4, 5, and 7), with or without ethanol, at room temperature or at 37°C (Appendix Table S1), without finding a condition where the morphology of the PFA-fixed samples consistently bettered that of glyoxal-fixed samples. We then proceeded to compare PFA and glyoxal fixation quantitatively. We first tested the speed with which these fixative solutions penetrate the cell membrane, by monitoring the fluorescence of propidium iodide, a fluorogenic probe that binds nucleic acids, and cannot enter living cells (Davey & Kell, 1996). Paraformaldehyde fixation allowed propidium iodide entry into cultured cells only after ~40 min, while glyoxal was substantially faster (Fig 1A). The same was observed using the membrane-impermeant styryl dye FM 1-43 (Betz et al, 1992): Glyoxal fixation enabled significant FM 1-43 penetration within 1–2 min (Fig 1B). The difference in membrane penetration is probably due to the ethanol present in the glyoxal fixative, since the addition of ethanol to the PFA solution enhances its penetration into cells in a similar fashion (Appendix Fig S1), albeit it did not improve immunostainings with PFA (Appendix Fig S1; we would like to point out that low pH values, 4 and 5, also failed to improve PFA immunostainings, as shown in the same Appendix figure). In the same experiments, FM 1-43 addition enabled us to visualize endocytotic events that took place during PFA fixation. Such events could be observed in every fixed cell (Fig 1B) and indicated that the cells were still active during PFA fixation, from the point of view of membrane trafficking. No such events could be detected during glyoxal fixation. Figure 1. Comparison of cell penetration by PFA and glyoxal Speed of propidium iodide (PI) penetration into fibroblasts during 60 min of fixation with either 4% PFA or 3% glyoxal. N = 3 independent experiments. Glyoxal fixation enables PI to penetrate far more rapidly into the cells. Speed of FM 1-43 penetration in similar experiments. The arrowhead points to one example of ongoing endocytosis during PFA fixation. N = 3–4 independent experiments. The general pattern of FM 1-43 entry was similar to that of propidium iodide. Only the first 10 min are shown, to enable an optimal observation of the kinetics of the first stages of FM 1-43 entry. The results parallel those obtained with PI: faster penetration during glyoxal fixation. Data information: Scale bar = 40 μm; **P < 0.01 (two-sided Student's t-test). Download figure Download PowerPoint The hypothesis that cells were still partially active during PFA fixation, and less so in glyoxal fixation, was also confirmed by other experiments. First, we tested whether transferrin, which is readily endocytosed by a clathrin-mediated pathway, through the involvement of the transferrin receptor, is internalized during fixation. We applied fluorescently conjugated transferrin onto cells during fixation with glyoxal or PFA (Appendix Fig S2). We found that it was mainly fixed onto the plasma membrane by glyoxal, but that it was present both in the cells and on the membrane during PFA fixation (Appendix Fig S2). Second, we tested whether the acidic lumen of the lysosome was maintained after fixation, by applying the probe LysoTracker (Appendix Fig S3). Substantial LysoTracker labeling was observed after PFA fixation, but not after glyoxal fixation. Both of these experiments, therefore, indicate that glyoxal fixation stops cellular functions more efficiently than PFA. The higher speed of membrane penetration seen with glyoxal was coupled to a better preservation of the general cell morphology, as observed by imaging cells during fixation (Fig 2). Paraformaldehyde fixation was associated with the formation of membrane blebs and vacuoles, with organelle movement, and with a general change in the cell morphology (Fig 2). Glyoxal fixation appeared to modify the cell morphology far less. This impression was confirmed by calculating the correlation coefficient between the initial cell images and images acquired at 5-min intervals during fixation (Fig 2). To obtain a similar view at the level of single organelles, we imaged the movement of endosomes labeled with fluorescently conjugated transferrin or cholera toxin. As for the general cell morphology, glyoxal reduced the organelle movement more than PFA (Appendix Fig S4). Figure 2. A comparison of morphological changes taking place during fixation with PFA or glyoxalThe changes were visualized by DIC images taken at 5-min intervals during fixation. The graph shows the correlation of each image to the first frame. N = 50 (PFA) and 54 (glyoxal) cellular regions analyzed, from three independent experiments (mean ± SEM). The higher correlation value indicates that glyoxal preserves the initial cell morphology with higher accuracy than PFA. Scale bar = 20 μm; **P < 0.01 (two-sided Student's t-test). Download figure Download PowerPoint We also monitored the morphology of mitochondria, which are known to become fragile during fixation. We visualized mitochondria in living cells, by tagging them with a GFP-linked reporter (TOMM70, Appendix Fig S5), and imaged them again after fixation. Glyoxal preserved mitochondria at least as well as PFA. Moreover, ethanol addition to the PFA solutions worsened the preservation of mitochondria morphology, which suggests that ethanol does not improve PFA fixation, although it enhances its membrane penetration (Appendix Fig S5). To test this issue further, we analyzed the correlation between the pre- and post-fixation images for fluorescent protein chimeras of a mitochondria reporter (TOMM70), a Golgi apparatus reporter (GalNacT2), a plasma membrane reporter (SNAP25), a cytoskeleton reporter (tubulin), and a vesicular reporter (synaptophysin). The correlations were similar among the two fixatives for TOMM70, GalNacT2, tubulin, and SNAP25. However, the pre- and post-fixation correlations in glyoxal fixed samples were higher for synaptophysin (Appendix Fig S6), which marks the most mobile elements we investigated in this experiment (vesicles). We then tested the protein cross-linking capacity of the different fixatives, by monitoring the proportion of the proteins that remained unfixed. We incubated brain cytosol samples with different fixatives for 60 min and followed this by running the samples on polyacrylamide gels (Fig 3A, Appendix Fig S7). Paraformaldehyde, with or without ethanol addition, left ~40% of the proteins unaffected (unfixed). Glyoxal (both pH 4 and 5) reduced this unfixed pool to ~20%. Shorter fixation times reduced the amount of fixed proteins for all fixation conditions (Appendix Fig S7). Glyoxal, both at pH 4 or at pH 5, fixed more proteins than PFA, PFA and ethanol or PFA at low pH, at all time points (Appendix Fig S7). Figure 3. Comparison of protein and RNA fixation by PFA and glyoxal SDS–PAGE gel showing rat bra