Anti doublecortin dcx

The sections (10-μm) were incubated in blocking solution with primary antibodies: anti-nestin (1:400; MAB353, Millipore, Billerica, MA, USA) as a marker of neural stem cells (NSCs) and neural progenitor cells (NPCs), anti-glial fibrillary acidic protein (GFAP) (1:500; No. 3670, Cell Signaling Technology, MA, United States) as a marker of actively dividing astrocytes, anti-neurofilament 200 (NF-200; 1:2000; Sigma) to label neuronal axons, anti-NeuN (1:800; MAB377; Millipore) as a marker of mature neurons, anti-doublecortin (DCX; 1:1000, 4604S, Cell Signaling Technology), a microtubule-associated protein primarily expressed by migratory immature neurons and neuronal neural progenitors, is widely used as a marker of adult neurogenesis, anti-CD68 (Cell Signaling Technology) as a marker of macrophages. Alexa Fluor 594-conjugated or Alex Fluor 488-conjugated secondary antibodies were used for detection. Nuclei were counterstained with 25 μg/mL DAPI (Sigma-Aldrich). Images were taken by a fluorescence microscope (Eclipse 80i, Nikon) equipped with a high-resolution color digital camera (Digital Sight US-U2, Nikon) The results were analyzed using Image-Pro Plus software (Media Cybernetics, Inc.).

PFA-fixed brain hemispheres were processed as previously described [23 (link)] with minor modifications. The 50 µm coronal sections were incubated in anti-glial fibrillary acidic protein (GFAP) (Thermo Fisher Scientific, Waltham, MA, USA, 1:200), anti-ionized calcium-binding adaptor molecule 1 (Iba1) (Wako, Osaka, Japan, 1:2000) and anti-doublecortin (DCX) (Cell Signaling Technology 1:600) primary rabbit antibodies overnight at 4 °C. A biotinylated goat anti-rabbit secondary antibody (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, USA) was used for the incubation of free-floating sections at room temperature for 90 min to perform chromogenic IHC.

One-half of the rat brains were processed as previously described [33 (link)]. The sections were incubated in anti-ionized calcium-binding adaptor molecule 1 (Iba1) (Wako, Osaka, Japan), anti-glial fibrillary acidic protein (GFAP) (Thermo Fisher Scientific, Waltham, MA, USA) or anti-doublecortin (DCX) (Cell Signaling Technology) primary rabbit antibodies. A biotinylated goat anti-rabbit secondary antibody was used for chromogenic IHC.