Rabbit anti map2

Seeded NSCs were washed with PBS and fixed with ice-cold 4% paraformaldehyde for 15 min at room temperature. The cells were then permeabilized with 0.5% Triton X-100 in TBS for 5 min and blocked with 2% FBS in TBS for 30 min at room temperature. The cells were incubated with primary antibodies: rabbit anti-sox2 (1:200, cell signaling, CA, USA), mouse anti-nestin (1:200, Millipore, MA, USA), rabbit anti-MAP2 (1:200, Millipore, MA, USA), mouse anti-β-tubulin III (1:200, Millipore, MA, USA), rabbit anti-GFAP (1:200, Stem Cell Technologies, BC, CA), rabbit anti-phosphorylated IRF3 (Ser396) (1:200, cell signaling, CA, USA), or mouse anti-ZIKV E protein (1:500, GeneTex, CA, USA) at 4 °C overnight. The cells were then washed three times with TBS and incubated with Dylight 594-conjugated donkey anti-mouse secondary antibody or Dylight 488-conjugated goat anti-rabbit secondary antibody (1:1000, Jackson ImmunoResearch Laboratories, PA, USA) for 1 h at room temperature. The cells were then washed three times with TBS, and the cell nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich, MO, USA). The images were collected with a fluorescence microscope (Olympus BX51, Olympus, Tokyo, JP).

Western blot analysis was performed as described previously [35 (link)], with slight modifications. Briefly, cells were lysed on ice with radioimmunoprecipitation (RIPA) lysis buffer supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and sonicated to reduce sample viscosity. Protein concentration was determined using the bicinchoninic acid assay (Pierce, Rockford, IL, USA), equal amounts of protein were separated on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Western blotting was performed using the following antibodies at 4 °C overnight: rabbit anti-β-III-tubulin (1:5000; Covance); rabbit anti-MAP-2 (1:2000; Millipore); rabbit anti-GFAP (1:5000; Abcam); rabbit anti-CNPase, rabbit anti-JNK, rabbit anti-p-JNK, rabbit anti-c-Jun, rabbit anti-p-c-Jun^ser63, and rabbit anti-p-c-Jun^ser73 (all 1:1000; all from Cell Signaling Technology); and mouse anti-β-actin (1:10,000; Sigma-Aldrich). Blots were incubated with horseradish peroxidase-labelled secondary anti-rabbit and anti-mouse antibodies, and immunoreactive bands were visualized on film by enhanced chemiluminescent substrate (Pierce, Rockford, IL, USA) (all 1:10,000, Abcam). Western blots were quantified with ImageJ software from three independent experiments. The intensities of the bands were normalized to β-actin.

After DIV 12, hippocampal primary neurons were fixed for 30 min with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), washed with DPBS (Invitrogen) three times and then blocked with 5% normal donkey serum in 0.1% TBS-Triton (TBS-TX) buffer for 2 h at room temperature. Primary antibodies were diluted in blocking solution at 4°C using the following dilutions: 1:1000 rabbit anti-MAP2 (Millipore), 1:1000 mouse anti-MAP2 (Sigma), 1:1000 mouse anti-Synapsin-1 (Abcam), 1:500 rabbit anti-MeCP2 (Cell Signaling Technology) and 1:500 mouse anti-MeCP2 (Sigma). After incubation in primary antibodies overnight, coverslips were incubated in the appropriate secondary antibodies diluted in blocking solution for 2 h at room temperature.
The brain tissues were fixed 2 weeks after stereotaxic injection by vascular perfusion through the left ventricle of the heart with sequential delivery of 50 ml of saline and 60 ml of 4% paraformaldehyde in 0.1 M PB. Coronal brain sections (40 μm) were prepared and processed for immunostaining using the anti-DCX (Santa Cruz; 1:300) antibodies.