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

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.

Glyoxal Does Not Preserve Cellular Proteins as Accurately as PFA: A Microscopical Survey of Epitopes

“…In this study, we wanted to compare those two aldehyde fixatives side-by-side using super-resolution confocal microscopy to determine whether Gly, as a historic and lesser-known compound, has any superior capacity for fixation over cellular proteins. Very recently, Richter et al published a comprehensive and well-designed data series pertaining to Gly vs. PFA fixation and concluded that 51 cellular targets were better preserved after Gly fixation (Richter et al, 2018). This led us to test Gly for our samples, most of which are related to male and female reproductive organs, early embryonic and adult stem cells.…”

“…With suitable catalysts or other reaction accelerators, Gly forms two-carbon adducts with nearly all end groups in proteins and carbohydrates, leaving most of them unimpaired for subsequent immunohistochemical (IHC) demonstration. Due to the previously reported disadvantages of PFA, Gly has recently been suggested superior to PFA as being less toxic, and preserves cells better as investigated using super-resolution microscopy (Richter, Revelo et al, 2018). There are also a number of reports comparing the staining and IHC labelling results between Gly-and FA-fixed tissues.…”

RNA‐guided endonuclease – in situ labelling (RGEN‐ISL): a fast CRISPR/Cas9‐based method to label genomic sequences in various species

“…The application of unfixed nuclei resulted in the strongest RGEN-ISL signals, but was accompanied by destructed nuclei. Furthermore, the exclusive fixation of tissues in glyoxal only (Richter et al, 2018) did not perform better than formaldehyde (Fig. S4).…”

Circumvention of common labeling artifacts using secondary nanobodies

“…A more efficient fixative such as glutaraldehyde (GLU) could be used, but it generates unwanted autofluorescence and only few affinity molecules find their target epitopes after GLU crosslinking. A recently described di-aldehyde alternative that seems to alleviate some of these problems caused by PFA and GLU is glyoxal 36 , although its implementation is very recent and the vast majority of researchers still use PFA-fixation for conventional immunofluorescence.…”