A Quantitative Comparison of Medial and Coronal Dentate Gyrus Microdissection Strategies and a Softening-Based...

A reliable isolation of the dentate gyrus (DG) is a critical pre-analytical step for region-specific neurobiological assays, yet DG microdissection practices vary widely and are rarely compared quantitatively under standardized conditions. In addition, long-term paraformaldehyde-fixed archival brain tissue is commonly regarded as unsuitable for microdissection because of reduced pliability and poor anatomical contrast, limiting its use for training and protocol development. Here, we quantitatively compare two commonly used DG microdissection strategies, a medial (intact-block) approach and a coronal (slice-guided) approach across fresh, fixed, and softened-fixed rat brain hemispheres under matched conditions. To enable the use of archival material, fixed hemispheres were subjected to a simple 15-day slow-running tap water softening protocol to improve tissue handling and landmark visibility. Dissection duration and anatomical specificity were evaluated, the latter quantified by measuring residual cornu ammonis (CA)1-3 area on hematoxylin-eosin-stained coronal sections following DG removal. In fresh tissue, the medial approach enabled significantly faster DG isolation than the coronal approach, while both strategies achieved comparable anatomical specificity. In softened-fixed tissue, dissection times increased for both approaches, but the same relative performance ranking was preserved. Softening markedly improved tissue pliability and boundary visualization, particularly benefiting the coronal, stepwise dissection strategy. Residual CA1-3 areas did not differ significantly between approaches or tissue states. This study provides a validated, training-oriented DG microdissection workflow that supports methodological standardization, reproducibility, and 3R-aligned use of archival tissue, strengthening the pre-analytical foundation for downstream region-specific neuroscience assays.