Protocol to study adult neurogenesis in fresh-frozen human hippocampal tissue using an immunofluorescence quantitative approach

Adult hippocampal neurogenesis (AHN) results in the generation of new neurons throughout adulthood, thereby contributing to key hippocampal functions such as learning and mood regulation. Here, we present a protocol for the immunofluorescence-based detection of AHN markers in fresh-frozen human hippocampal sections. We describe steps for tissue fixation, antibody incubation, and autofluorescence elimination. We then detail procedures for image acquisition and quantification. This protocol allows the reliable identification of distinct cell populations and could be adapted for broader immunohistochemical applications. For complete details on the use and execution of this protocol, please refer to Márquez-Valadez et al. 1 • Protocol for immunostaining fresh-frozen postmortem human hippocampal tissue • Reliable detection of markers of adult hippocampal neurogenesis in human tissue • Provides guidance for acquiring confocal images and determining cell densities • Guidance for morphometric determinations and dentate gyrus vasculature analysis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Adult hippocampal neurogenesis (AHN) results in the generation of new neurons throughout adulthood, thereby contributing to key hippocampal functions such as learning and mood regulation. Here, we present a protocol for the immunofluorescence-based detection of AHN markers in fresh-frozen human hippocampal sections. We describe steps for tissue fixation, antibody incubation, and autofluorescence elimination. We then detail procedures for image acquisition and quantification. This protocol allows the reliable identification of distinct cell populations and could be adapted for broader immunohistochemical applications.