Comparative Analysis of Davidson and Glyoxal Fixatives on Autofluorescence and Immunolabeling in Medaka (Oryzias latipes) Tissues.

Background: Fixation influences the quality of staining across species, especially in neuroscience, where accurate visualization of neuronal structures and protein localization is crucial for understanding brain function and pathology. This study compared two commonly used fixatives-9% glyoxal (G-fix) and Davidson's solution (D-fix)-regarding their effects on autofluorescence, immunolabeling specificity, and histological quality in medaka brain tissue. Methods: Mixed-sex medaka from five strains were fixed with either G-fix or D-fix. Autofluorescence was assessed in posterior bodies and brain tissues, including sections stained with fluorescently conjugated secondary antibodies alone. Tissues were also injected with fluorescent dyes or immunolabeled for neuronal markers (PGP9.5, NeuN, and NCAM) using fluorescent secondary antibodies. Hematoxylin and eosin (H&E) staining and immunohistochemistry were used to evaluate tissue morphology and chromogenic antigen detection. Results: Both fixatives induced autofluorescence: D-fix enhanced blue signals, while G-fix increased green and red fluorescence. These autofluorescence levels were significantly weaker than those from fluorescent dyes or PGP9.5 immunolabeling. Posterior body tissue showed patterns similar to deparaffinized brain sections, supporting its use for pre-screening fixation. G-fix yielded more neuron-specific PGP9.5 staining, whereas D-fix showed broader signal distribution. NeuN and NCAM were not detected, likely due to antibody incompatibility. PGP9.5 was undetectable by immunohistochemistry, while D-fix provided superior H&E staining quality. Conclusions: Although both fixatives induced autofluorescence, their signals were weaker than those of conventional dyes and antibodies. Glyoxal improved specificity for neuronal immunofluorescence, while Davidson enhanced histological detail. These findings provide practical guidance for optimizing fixation strategies in medaka-based neuroscience and histopathological research.