a–k, Effects of fixation time. a, Schematic diagram of the experimental design. Briefly, a hippocampal fragment from four control subjects (61–87 years of age; Extended Data Fig. 1) was dissected and cut into 0.5-cm-thick blocks along the rostro–caudal axis of this structure. Each block was fixed for a different period of time in 4% PFA at 4 °C. b–f, Influence of fixation time on the visualization of DCX staining in the human DG. b, Number of DCX+ cells detected after different fixation times (one-way ANOVA, F 8,32 = 23.90, P < 0.0001). The graph represents mean values ± s.e.m. c–f, Representative images of DCX+ cells, showing the differences in signal quality and background intensity caused by different fixation times. Weak tissue preservation, high-intensity background, and low intensity and specificity of the signal were observed after 1 h of fixation in 4% PFA. In contrast, 2–12 h of fixation in 4% PFA rendered a good signal/background ratio, although tissue robustness was minimal. In contrast, 24 and 48 h of fixation almost abolished DCX detection and substantially increased nonspecific background. Thus, given the marked decay in DCX signal after 12 h of fixation, samples fixed for 24 and 48 h were subjected to aldehyde elimination with 0.5% NaBH 4 and to a HC-AR protocol, which revealed the presence of thousands of DCX+ cells in the adult human DG. Moreover, NaBH 4 and HC-AR allowed unambiguous identification of neuronal characteristics in these cells. g–k, Influence of fixation time on the visualization of PSA-NCAM staining in the human DG. g, Number of PSA-NCAM+ cells detected (one-way ANOVA, F 8,32 = 16.54, P < 0.0001). The graph represents mean values ± s.e.m. h–k, Representative images of PSA-NCAM staining in each fixation condition tested. Fixation times longer than 12 h impeded PSA-NCAM detection, and incubation with NaBH 4 + HC-AR was necessary to allow stereological estimation of the density of PSA-NCAM+ cells. These histological pretreatments allowed identification of thousands of PSA-NCAM+ cells in the DG. In a–k, n = 4 control subjects. 10–20 measurements were performed for each subject. l–v, Effects of formalin fixation on DCX and PSA-NCAM detection in the DG. l, Experimental design. Briefly, the hippocampi of two control subjects (control 2 and control 4; Extended Data Fig. 1) were dissected, and two 0.5-cm-thick blocks per subject were obtained. One of these blocks was fixed in 4% PFA for 24 h, whereas the other block was stored in 3.7% formalin for 6 months. m–p, Representative images of DCX staining in the different experimental conditions. q, Number of DCX+ cells detected in samples fixed either in 4% PFA for 24 h or in 3.7% formalin for 6 months, with or without NaBH 4 + HC-AR pretreatment. The graph represents mean values. NaBH 4 + HC-AR steps increase the number of DCX+ cells detected in samples fixed with both PFA and formalin. However, neither the intense background nor the marked decay in DCX signal caused by formalin fixation was counteracted by NaBH 4 + HC-AR. r–u, Representative images of PSA-NCAM staining in the different experimental conditions. v, Number of PSA-NCAM+ cells detected in samples fixed either in 4% PFA for 24 h or in 3.7% formalin for 6 months, with or without NaBH 4 + HC-AR. The graph represents mean values. NaBH 4 + HC-AR increased the number of PSA-NCAM+ cells detected in samples fixed with PFA. However, formalin fixation abolished the PSA-NCAM signal, and this effect was not prevented by NaBH 4 + HC-AR. In l–v, n = 2 control subjects. 10–20 measurements were performed for each subject. Yellow scale bars, 50 μm. Green triangles indicate DCX+ cells; red triangles indicate PSA-NCAM+ cells. In b and g, asterisks indicate significant differences with respect to the samples fixed for 1 h in Tukey post hoc comparisons. *0.05 > P ≥ 0.01; **0.01 > P ≥ 0.001; ***P < 0.001.