A double immunolabeling technique was employed to investigate the specific cell subpopulations involved in proliferation. The procedures were previously described and consist of an initial incubation with blocking and permeabilization solutions for 60 min each (Borroto-Escuela et al., 2021 (link), 2022 (link); Mirchandani-Duque et al., 2022 (link)). Pairs of primary antibodies mouse anti-PCNA (1:1500, P8825, Sigma, St. Louis, MO, USA)/rabbit anti-DCX (Abcam, ab18723, 1:2000) or mouse anti-PCNA (1:1500, P8825, Sigma, St. Louis, MO, USA)/rabbit anti-GFAP (Abcam, ab7260, 1:1500) were used to incubate the sections for 24 h at 4°C. Subsequently, incubations were performed with proper secondary antibodies: Donkey anti-mouse AlexaFluor 488 (Abcam, ab150105, 1:200) and Donkey anti-rabbit AlexaFluor 647 (Abcam, ab150075, 1:200). The sections were mounted with a fluorescent mounting medium containing DAPI (4’,6-diamidino-2-phenylindole) for nuclei detection (Abcam, ab104139). Quantitative analysis of the PCNA/DCX- and PCNA/GFAP-immunostained cells in the dentate gyrus of the dorsal hippocampus was conducted as described (Cohen et al., 2018 (link); Mirchandani-Duque et al., 2022 (link)).
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