# Evaluation of Glyoxal fixation for immunohistochemistry of the retina

## TL;DR

This study evaluated glyoxal as a fixative for retinal immunohistochemistry but found it less effective than formaldehyde.

## Contribution

The study provides a detailed comparison of glyoxal and formaldehyde fixation for retinal immunohistochemistry in various tissue preparations.

## Key findings

- Glyoxal fixation produced fragile retinas unsuitable for consistent wholemount dissection.
- Formaldehyde generally provided better or equivalent signal-to-background immunolabelling compared to glyoxal.
- No significant improvement in immunohistochemical signals was observed when glyoxal replaced formaldehyde.

## Abstract

Immunohistochemistry has become an essential tool in retinal research. Formaldehyde is the gold standard fixative, but the development of an improved fixative is of keen interest. Herein, we performed a comprehensive evaluation of the compatibility of glyoxal fixation with retinal immunohistochemistry, using wholemounts, cryosections and paraffin-embedded eyes. Immunohistochemistry was performed in normal rat eyes, and, to facilitate localisation of stress-response proteins, eyes subjected to laser-induced retinal injury or injected with the endotoxin lipopolysaccharide. Regarding wholemounts, glyoxal fixation produced retinas that were too fragile to be consistently dissected as pristine wholemounts. In terms of antigenicity, we observed no consistent improvement when glyoxal fixation was used. Some antibodies produced higher signal intensities, but a greater number displayed weaker signal-to-background patterns of labelling compared to formaldehyde fixation. For cryosections and paraffin sections, we likewise found no compelling evidence that immunohistochemical signals were intensified when formaldehyde was replaced by glyoxal. For the 50 antibodies tested, formaldehyde typically produced signal-to-background immunolabelling that was equivalent or superior to glyoxal. In conclusion, the results of this study do not support the use of glyoxal fixation for immunohistochemistry of the rat retina, but with the caveat that improved formulations and protocols may address the limitations exposed herein.

The online version contains supplementary material available at 10.1038/s41598-025-04226-7.