Glyoxal as an alternative fixative to formaldehyde in immunostaining and super‐resolution microscopy |
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| Authors: | Katharina J Seitz Martin S Helm Deblina Sarkar Rebecca S Saleeb Elisa D'Este Jessica Eberle Eva Wagner Christian Vogl Diana F Lazaro Frank Richter Javier Coy‐Vergara Giovanna Coceano Edward S Boyden Rory R Duncan Stefan W Hell Marcel A Lauterbach Stephan E Lehnart Tobias Moser Tiago F Outeiro Peter Rehling Blanche Schwappach Ilaria Testa Bolek Zapiec Silvio O Rizzoli |
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| Institution: | 1. Department of Neuro‐ and Sensory Physiology, University of G?ttingen Medical Center, G?ttingen, Germany;2. International Max Planck Research School Molecular Biology, G?ttingen, Germany;3. MIT Media Lab;4. Edinburgh Super‐Resolution Imaging Consortium, Institute of Biological Chemistry, Biophysics, and Bioengineering, Heriot‐Watt University, Edinburgh, UK;5. Department of NanoBiophotonics, Max‐Planck‐Institute for Biophysical Chemistry, G?ttingen, Germany;6. Department of Neural Systems, Max‐Planck‐Institute for Brain Research, Frankfurt am Main, Germany;7. Heart Research Center G?ttingen, Department of Cardiology & Pulmonology, University Medical Center G?ttingen, G?ttingen, Germany;8. German Center for Cardiovascular Research (DZHK) Site G?ttingen;9. Institute for Auditory Neuroscience and InnerEarLab, University Medical Center G?ttingen, G?ttingen, Germany;10. Max‐Planck‐Institute for Experimental Medicine, Auditory Neuroscience Group, G?ttingen, Germany;11. Department of Experimental Neurodegeneration, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Center for Biostructural Imaging of Neurodegeneration, University Medical Center G?ttingen, G?ttingen, Germany;12. Max‐Planck‐Institute for Experimental Medicine, G?ttingen, Germany;13. Department of Cellular Biochemistry, University Medical Center G?ttingen, G?ttingen, Germany;14. Department of Molecular Biology, University Medical Center G?ttingen, G?ttingen, Germany;15. Department of Applied Physics and Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden;16. Departments of Brain and Cognitive Science and Biological Engineering, MIT Media Lab and McGovern Institute, Cambridge, MA, USA;17. Max‐Planck‐Institute for Biophysical Chemistry, G?ttingen, Germany;18. Max Planck Research Unit for Neurogenetics, Frankfurt am Main, Germany;19. Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, G?ttingen, Germany |
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| 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. |
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| Keywords: | fixation glyoxal immunocytochemistry
PFA
super‐resolution Microscopy |
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