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

Year: 2026

Type: article

Abstract: 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.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.104344

Authors: Marta Gallardo-Caballero, Berenice Márquez-Valadez, María Llorens-Martín

Institutions: Consejo Superior de Investigaciones Científicas, Biomedical Research Networking Center on Neurodegenerative Diseases, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid

Language: English

Topic: Neurogenesis and neuroplasticity mechanisms

Subfield: Developmental Neuroscience

Field: Neuroscience

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $1,550