Prolonged fixation and post-mortem delay impede the study of adult neurogenesis in mice

Adult hippocampal neurogenesis (AHN) gives rise to new neurons throughout life. This phenomenon takes place in more than 120 mammalian species, including humans, yet its occurrence in the latter was questioned after one study proposed the putative absence of neurogenesis markers in the adult human hippocampus. In this regard, we showed that prolonged fixation impedes the visualization of Doublecortin+ immature neurons in this structure, whereas other authors have suggested that a dilated post-mortem delay (PMD) underlies these discrepancies. Nevertheless, the individual and/or additive contribution of fixation and the PMD to the detection (or lack thereof) of other AHN markers has not been studied to date. To address this pivotal question, we used a tightly controlled experimental design in mice, which allowed the dissection of the relative contribution of the aforementioned factors to the visualization of markers of individual AHN stages. Fixation time emerged as the most prominent factor globally impeding the study of this process in mice. Moreover, the visualization of other particularly sensitive epitopes was further prevented by prolonged PMD. These results are crucial to disambiguate current controversies related to the occurrence of AHN not only in humans but also in other mammalian species. Significant impact of post-mortem time and fixation method on immunohistochemistry performance of antibody-based staining of common markers relevant to adult neurogenesis.