Optimizing fixation of nonhuman primate testis for integrated histopathology and RNA-based molecular pathology

Emerging molecular pathology platforms, including spatial transcriptomics and RNA-based in situ assays, now permit simultaneous characterization of tissue morphology and gene expression from the same tissue section. These technologies challenge traditional fixation paradigms in toxicologic pathology, which have historically prioritized morphological preservation over nucleic acid integrity. The Society for Toxicologic Pathology recommends modified Davidson's Fixative (mDF) for evaluation of mammalian testicular tissue because of its superior preservation of tissue architecture; however, its effects on RNA quality are less well characterized. This study evaluated the histomorphological quality and RNA quality in dissected non-human primate (NHP) testis samples preserved using different fixation methods. mDF was confirmed to better preserve NHP testis morphology compared to 4% PFA. 4% PFA improved preservation of larger RNA fragments compared to mDF; however, the effects of mDF-based RNA degradation could be rescued by a shorter fixation time. Ultimately, one must weigh the advantages and disadvantages of using mDF versus 4% PFA to fix dissected NHP testis samples depending on the intended downstream applications of the sample. For purely histological applications, researchers should use mDF, and for purely molecular assays, 4% PFA is the preferred fixative. For applications that require both histological and molecular optimization, mDF fixation for a shorter duration (e.g. 12 hours) or 4% PFA fixation for a longer duration (e.g. 24 or 48 hours) should be considered.