A freeze-substitution approach with solvent-based glyoxal fixative to prevent distortion of ocular structures.

Investigating the function of delicate mammalian eyes often requires chemical fixation, histological sectioning, immunohistochemistry (IHC) and in situ hybridization (ISH). One of the long-standing challenges in the ocular histology field is the limited success of maintaining intact morphology via cryo- or paraffin procedures. Although our latest protocol significantly improved the morphology of mouse eyeball sections, the window technique is time-consuming and requires extensive practice to avoid damage while making windows. In this study, we present a novel glyoxal fixative that is suitable for a freeze-substitution approach to improve both morphology and molecular target preservation of mouse eyes. The method prevents morphology distortion in all tested eyeballs. Therefore, it suits a variety of research needs from morphological examination to investigation of single-molecule RNA expression, using hematoxylin and eosin (H&E) stain, IHC, and ISH assays on either frozen (cryo) or paraffin-infiltrated tissue sections. In addition, this method can be easily performed in many histology laboratories.