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. 2021 Aug 26;22(17):9225. doi: 10.3390/ijms22179225

Figure 3.

Figure 3

Effect of an acute administration of KET/MEL combination on the doublecortin and K167 expression in the mice hippocampus. Animals (n = 3 per group) were i.p injected with the VEH or a single dose of MEL (4 mg/kg), KET (1.5 mg/kg), or the combination of KET/MEL (1.5/4 mg/kg) at ZT 18, 30 min before the FST. Then, they were anesthetized and perfused as described in methods. The brains were sectioned in 30 µm slices and immunostained with an anti-doublecortin antibody and a secondary DyLight 488 antibody. Nuclei were stained with DAPI (A–D). The number of doublecortin-positive cells were counted in the subgranular zone in the dentate gyrus (E), each point representing a determination in one brain slice image. A total of 18 slice images (two images from two different hippocampal zones) derived from 9 brain slices of 3 mice per group were assessed. Data was analyzed using a one-way ANOVA, F(3,25) = 263.3, p = 0.003. Another mice group (n = 4 for DCX and n = 3 for Ki67, per group) was similarly injected and after the FST they were sacrificed. The hippocampal region was dissected, homogenized, and analyzed by Western blot. Doublecortin and Ki67 bands were immunostained with specific antibodies and detected by secondary DyLight 680 antibodies. Quantitative graphs for doublecortin (DCX) and Ki-67 are shown in panels (F,G), respectively. Samples were run for duplicate. Each point represents two technical replicates. Data were analyzed using a one-way ANOVA followed by a Tukey post-test. Data were normalized by total protein as well as by maximal and minimal values. (F(3,12) = 12.8, p = 0.0005 for DCX and F(3,8) = 13.35, p = 0.02 for Ki67). * p < 0.05, *** p < 0.001. Scale bar = 100 µm).