Improvement and development of immersion fixatives for enhanced tissue preservation in the whole eyeball specimens

The wall of eyeball limits the penetration of the common fixatives, raising inadequate fixation or degeneration of internal tissue and posing challenges for preparing thin sections of the whole eyeball. In addition, many formaldehyde-containing fixatives exhibit the strong cross-linking effect that can excessively mask antigenic epitopes, thereby hampering immunological analyses. This study tried to develop an available fixative that exerts high permeability to mouse whole eyeballs and applicability to immunostaining. Glyoxal is known to have milder cross-linking capacity compared to formaldehyde, bringing better preservation of epitopes. We substituted the formaldehyde component in Davidson's solution with glyoxal and supplemented 1-butanol, methanol, 2-mercaptoethanol, and tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl). Using modified fixatives and the GBMT solution, which was named by the initial letters of each component listed above, mouse eyeballs were immersion-fixed with the modified fixatives, and both tissue morphology and immunostaining quality were evaluated. The results showed that the GBMT solution prepared with glyoxal and specific combinations of additives improved morphological preservation of the eyeball tissues and enhanced immunofluorescence signals for certain cytoplasmic antigens, compared to the standard Davidson's solution. Our findings demonstrated that the successful development of a novel fixative enables both high-quality whole eyeball sectioning and improved immunostaining performance through immersion fixation alone.