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

Katharina N. Richter, Natalia H. Revelo, Katharina J. Seitz, Martin S. Helm, Deblina Sarkar, Rebecca Sonia 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 MoserTiago Outeiro, Peter Rehling, Blanche Schwappach, Ilaria Testa, Bolek Zapiec, Silvio O. Rizzoli

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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.

Original languageEnglish
Article numbere201695709
JournalEMBO Journal
Issue number23
Early online date16 Nov 2017
Publication statusPublished - 1 Dec 2017


  • Journal Article


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