Rabbit anti doublecortin dcx

The sections were processed under the same conditions to obtain comparable immunohistochemistry among groups as described in a previous study (Jung et al., 2019). Three sections from 1.82 to 2.32 mm posterior to the bregma according to a mouse atlas (Paxinos & Franklin, 2001), separated by intervals of 150 μm, were obtained from each animal. Each tissue section was sequentially treated with 0.3% H₂O₂ in PBS for 30 min and 10% normal goat serum in 0.1 M PBS for 30 min at 25°C. The sections were first incubated overnight with rabbit anti‐Ki67 (1:1,000; Abcam), rabbit anti‐doublecortin (DCX, 1:2,000, Abcam), rabbit anti‐glucose transporter 3 (GLUT3; 1:50, Santa Cruz Laboratory), or rabbit anti‐phosphorylated cAMP response element‐binding protein at Ser133 (pCREB, 1:400; Cell Signaling) at 25°C. The next day, the sections were treated with biotinylated goat anti‐rabbit IgG (1:200; Vector) for 2 hr at 25°C. Subsequently, the sections were treated with streptavidin–peroxidase complex (1:200; Vector) for 2 hr at 25°C. Thereafter, the brain sections were visualized by reaction with 3,3′‐diaminobenzidine tetrahydrochloride (DAB, Sigma) in 0.1 M Tris‐HCl buffer (pH 7.2) and mounted on gelatin‐coated slides. Sections were dehydrated with graded concentrations of alcohol and mounted in Canada balsam (Kanto Chemical).

hNPCs were fixed, washed, and blocked as previously described13 (link), being followed by a 1 h incubation (at 37 °C) with primary antibodies: nestin (1:200, Millipore, Billerica, MA, USA) to detect neural progenitors, rabbit anti-doublecortin (DCX; 1:500, Abcam, Cambridge, MA, USA) to detect immature neurons, mouse anti-rat microtubule associated protein-2 (MAP-2; 1:500, Sigma) and neuronal nuclei (NeuN; 1:100, Millipore) to detect mature neurons. Secondary antibodies namely, Alexa Fluor 488 goat anti-rabbit immunoglobulin G (IgG, Life Technologies) and Alexa Fluor 594 goat anti-mouse immunoglobulin G (IgG, Life Technologies) were used for 1 h at 37 °C and then 10 min with 4-6-diamidino-2-phenylindole-dihydrochloride (DAPI; Life Technologies). Samples were observed under an Olympus BX-61 Fluorescence Microscope (Olympus, Hamburg, Germany). For this purpose, three coverslips and ten representative fields per condition were chosen and analyzed. In order to normalize the data between the different sets, the results are presented in percentage of cells. This was calculated by counting the cells positive for NeuN/MAP-2/DCX markers, and dividing this value by the total number of cells/field (DAPI-positive cells; n = 3).

Following incubation with secretome, the induction of differentiation was evaluated. Therefore, cells were fixed with 4% PFA (PanReac, Barcelona), washed with 1× PBS, and blocked as already described [28 (link)]. Following blocking, cells were incubated for 1 h at RT with the primary antibodies, namely, rabbit anti-doublecortin (DCX; 1:300, Abcam, USA), to detect immature neurons and mouse microtubule-associated protein-2 (MAP-2; 1:500, Sigma) and to detect mature neurons. After a washing step, cells were incubated for 1 h at RT with secondary antibodies (1:1000): Alexa Fluor 488 goat anti-rabbit (Thermo Fisher Scientific) and Alexa Fluor 594 goat anti-mouse (Thermo Fisher Scientific). Following this procedure, DAPI (Life Technologies) was added for 5 min. Coverslips were observed under an Olympus BX-61 Fluorescence Microscope (Olympus, Tokyo, Japan). Briefly, four coverslips per condition and ten representative fields per coverslip were analyzed, and the experiment was repeated independently four times. Results are presented as percentage of cells positive for MAP-2 or DCX markers divided by the total number of cells/field (DAPI-positive cells).