Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy PMID: 29146773

GLP-1 and Obesity Research

2026-08-18

Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy

EMBO J. 2018 Jan 4;37(1):139-159. doi: 10.15252/embj.201695709.

Katharina N Richter ¹ ² , Natalia H Revelo ¹ , Katharina J Seitz ¹ ³ , Martin S Helm ¹ ³ , Deblina Sarkar ⁴ , Rebecca S Saleeb ⁵ , Elisa D'Este ⁶ , Jessica Eberle ⁷ , Eva Wagner ⁸ ⁹ , Christian Vogl ¹⁰ ¹¹ , Diana F Lazaro ¹² ¹³ , Frank Richter ³ ¹⁴ , Javier Coy-Vergara ¹⁵ , Giovanna Coceano ¹⁶ , Edward S Boyden ¹⁷ , Rory R Duncan ⁵ , Stefan W Hell ⁶ , Marcel A Lauterbach ⁷ , Stephan E Lehnart ⁸ ⁹ , Tobias Moser ¹⁰ ¹¹ , Tiago F Outeiro ¹² ¹³ , Peter Rehling ¹⁴ ¹⁸ , Blanche Schwappach ¹⁵ , Ilaria Testa ¹⁶ , Bolek Zapiec ¹⁹ , Silvio O Rizzoli ²⁰ ²

Affiliations

1. Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany.
2. Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Göttingen, Germany.
3. International Max Planck Research School Molecular Biology, Göttingen, Germany.
4. MIT Media Lab.
5. Edinburgh Super-Resolution Imaging Consortium, Institute of Biological Chemistry, Biophysics, and Bioengineering, Heriot-Watt University, Edinburgh, UK.
6. Department of NanoBiophotonics, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
7. Department of Neural Systems, Max-Planck-Institute for Brain Research, Frankfurt am Main, Germany.
8. Heart Research Center Göttingen, Department of Cardiology & Pulmonology, University Medical Center Göttingen, Göttingen, Germany.
9. German Center for Cardiovascular Research (DZHK) Site Göttingen.
10. Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany.
11. Max-Planck-Institute for Experimental Medicine, Auditory Neuroscience Group, Göttingen, Germany.
12. 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.
13. Max-Planck-Institute for Experimental Medicine, Göttingen, Germany.
14. Department of Cellular Biochemistry, University Medical Center Göttingen, Göttingen, Germany.
15. Department of Molecular Biology, University Medical Center Göttingen, Göttingen, Germany.
16. Department of Applied Physics and Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden.
17. Departments of Brain and Cognitive Science and Biological Engineering, MIT Media Lab and McGovern Institute, Cambridge, MA, USA.
18. Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
19. Max Planck Research Unit for Neurogenetics, Frankfurt am Main, Germany.
20. Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Göttingen, Germany [email protected].

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.

Keywords

PFA; fixation; glyoxal; immunocytochemistry; super‐resolution Microscopy.