Standardizing dentate gyrus isolation for molecular adult hippocampal neurogenesis studies: a comparative and tissue softening-enabled workflow

Abstract Adult hippocampal neurogenesis (AHN) studies depend on dentate gyrus (DG) tissue isolated with high anatomical fidelity and controlled pre-analytical steps. To our knowledge, the two widely used DG microdissection approaches, medial (intact-block) and coronal (slice-guided) have not been directly compared under matched conditions, and the value of a simple tissue-softening step for operational standardization has not been quantified.To provide a comparative, quantitative validation of the medial and coronal DG microdissection approaches and to establish a tissue softening enabled workflow that laboratories can adopt for training and standardization.Adult rat hemispheres were assigned to seven groups (n = 3 hemispheres/group): fresh-medial, fresh-coronal, fixed-medial, fixed-coronal, softened-medial, softened-coronal, and intact control (fixed). A 15-day slow running-water rinse softened archival tissue. Outcomes were (i) operational performance (time to isolate DG) and (ii) anatomical specificity (residual CA1-CA3 area on H&E after the DG removal).In fresh tissue, the medial approach isolated DG in 51.7 ± 6.5 vs 125.3 ± 8.5 s for the coronal approach (~ 2.4× difference). In softened fixed tissue, both approaches were slower, but the speed ranking (medial < coronal) was preserved. Anatomical specificity did not differ among groups (residual CA1-CA3; one-way ANOVA, p > 0.05). Softening improved border visibility and handling, particularly for the coronal approach, providing an operational surrogate for training and standardization.Under matched conditions, medial approach offers faster procurement while coronal maximizes border visibility; both maintain comparable anatomical specificity. The 15-day softening protocol supports 3R-aligned training and standardization.