Sc 271390

Paraffin embedded slices were deparaffined and antigen retrieval carried out by incubating the samples in a 10 mM sodium citrate solution at pH 6 + 0.05% Tween20 in distilled water, where they were boiled 3 times, maintained at 95–98 °C for 20 min and finally cooled at room temperature. After blocking in 5% bovine serum albumin buffer, brain samples were incubated overnight with the following primary antibodies: mouse anti-GFAP (1:100, MA5-12023, Thermo Fisher) to label radial-glia/neural stem cells and then assess neurogenic activity close to the ventricular wall; mouse anti-DCX (doublecortin,1:50, sc-271390, Santa Cruz Biotechnology) to identify young neurons/neuroblasts; and mouse anti-Ki67 (1:50, STJ96966, St Johns Labs, UK) to label dividing cells/proliferation. To study the proliferation of neural stem/progenitor cells, we performed a double immunohistochemistry with Ki67 and Sox2 (1:100, Santa Cruz Biotechnology) for cell colocalization. The day after, brain samples were incubated with Alexa Fluor 488 and/or Texas Red secondary antibodies (both 1:300, Thermo Fisher) for 1 h at room temperature in the dark, and, after several washes, slices were mounted (F4680, Sigma). Negative controls received identical treatment except for omission of primary antibodies and showed no specific staining.

Brain tissues were cut into frozen sections. The frozen sections were air-dried at room temperature, washed with PBS, permeabilized with 0.4% Triton X-100, and submitted to antigen retrieval as for immunohistochemistry. After washing with PBS, sections were blocked in normal goat serum for 1 h. Then, the sections were incubated overnight at 4 °C with the following primary antibodies: anti-ALK5 (1:50, SAB4502958, Sigma-Aldrich, USA), anti-neuron-specific beta-III tubulin (1:50, MAB1195, R&D, USA), anti-GFAP (1:100, BM0055, Boster, China), anti-nestin (1:100, ab11306, Abcam, USA), and anti-DCX (1:50, sc-271390, Santa Cruz Biotechnology, USA) antibodies. The next day, the sections were washed with PBS and incubated with a mixture of goat anti-rabbit IgG-CFL 488 (1:100, sc-362262, Santa Cruz Biotechnology, USA) and goat anti-mouse IgG-CFL 555 (1:200, sc-362267, Santa Cruz Biotechnology, USA) at 37 °C for 1 h in the dark. Then, the sections were counterstained with DAPI for nuclei. Images were captured by a confocal laser scanning microscope (A1 + R, Nikon, Tokyo, Japan).

Mice were perfused transcardially with ice-cold 0.9% saline followed by ice-cold 4% paraformaldehyde solution (#G1101, Servicebio, Wuhan, Hubei, China). The whole brains were then removed, post-fixed in 4% paraformaldehyde overnight. Then the brains were washed in fluid water, dehydrated in ethanol and xylene, and embedded in melted paraffin for 2 h. After coagulation in room temperature, the brains were cut at 5 μm. Prior to immunofluorescence, the slices were deparaffined in xylene and dehydrated in ethanol. Approximately 10 sections, including the hippocampus region, were collected from each brain. After blocked with 3% BSA buffer, slices were incubated with antibody against DCX (#sc-271390, Santa Cruz, Dallas, TX, USA) at 4 °C overnight, and then in the second antibody for 1 h at room temperature. Nuclei were stained by DAPI (Ruchuang, Shanghai, China) for 15 min at room temperature. Fluorescent image acquisition was conducted using an inverted fluorescence microscope (Carl Zeiss, Germany). The data were analyzed by ImageJ Software.

The following primary antibodies were used for immunofluorescence staining: goat anti-tdTomato (1:500; catalog #AB8181-200, SICGEN; RRID:AB_2722750); rabbit anti-TRP73 (1:500; catalog #ab40658, Abcam; RRID:AB_776999); mouse anti-RELN (1:500; catalog #MAB5364, MilliporeSigma; RRID:AB_1293544); mouse anti-RELN (1:500; catalog #ab78540, Abcam; RRID:AB_1603148); rabbit anti-CALB1 (1:500; catalog #CB38, Swant; RRID:AB_10000340); mouse anti-DCX (1:25; catalog #sc-271390, Santa Cruz Biotechnology; RRID:AB_10610966); and goat anti-EGFP/YFP (yellow fluorescent protein; 1:500; catalog #AB0020-500, SICGEN; RRID:AB_2333100). The secondary antibodies used were as follows: donkey anti-goat Alexa Fluor 555 (1:1000; catalog #A21432, Thermo Fisher Scientific; RRID:AB_2535853); donkey anti-rabbit Alexa Fluor 488 (1:1000; catalog #A21206, Thermo Fisher Scientific; RRID:AB_2535792); and donkey anti-mouse Alexa Fluor 647 (1:1000; catalog #A31571, Thermo Fisher Scientific; RRID:AB_162542).

5-μm slices from P14 brain samples were deparaffinized, rehydrated and antigen retrieval was performed using a sodium citrate solution (10 mM sodium citrate + 0.05% Tween20 in distilled water at pH 6) where samples were boiled three times before keeping at 95–98 °C for 20 min. Once cooled at room temperature, the endogenous peroxidase was blocked with 3% H₂O₂ in PBS and slices were washed in PBS and incubated in a blocking solution (5% bovine serum albumin + 0.4%Triton X-100 in PBS) for 1 h. After several washes in PBS, brain sections were incubated with primary antibody to Ki67 (1:50; BD Pharmingen; #550609) or DCX (1:50; Santa Cruz Biotechnology; sc-271390) at 4 °C overnight.
The following day, slices were washed three times in PBS and incubated with secondary antibodies biotin conjugate (goat anti-mouse IgG H+L, #31800, Invitrogen, Thermo Fisher, USA) for 1 h at room temperature, followed by three washes in PBS and final incubation with horseradish peroxidase-streptavidin conjugate (1:500, 43-4323, Thermo Fisher, USA) for 30 min and posterior diaminobenzidine revealed. Finally, brain sections were counterstained with hematoxylin and mounted with DPX. Negative controls received identical treatment except for the omission of primary antibodies and showed no specific staining.

Brain slices for fluorescence immunohistochemistry were managed as detailed in the “Immunohistochemistry” section until incubation with primary antibody. For single labeling, radial-glia/neural stem cells were identified using an anti-glial fibrillary acidic protein (GFAP) antibody to assess neurogenic activity close to the ventricular wall (mouse anti-GFAP, 1:100, MA5-12023, Thermo Fisher, United States); an anti-doublecortin (DCX) antibody was used to identify young neurons/neuroblasts (mouse anti-DCX, 1:50, sc-271390, Santa Cruz Biotechnology, United States); cell proliferation was identified by an anti-Ki67 antibody (mouse anti-Ki67; 1:50, STJ96966, St Johns Labs, United Kingdom). Double immunohistochemical staining was used to detect the cellular co-localization of Ki67 with Sox2 (neural stem/progenitor cells; 1:100, Santa Cruz Biotechnology, United States). Immunoreactivity was revealed using Alexa Fluor 488 and Texas Red (1:300, Thermo Fisher, United States) secondary antibodies incubated in the dark for 1 h at room temperature. After final washes, fluoromount aqueous mounting medium (F4680, Sigma) was added, and each section was covered by a cover slip. Negative controls received identical treatment except for the omission of primary antibodies and showed no specific staining.