Fitc conjugated goat anti rabbit

The expression of BDNF, cleaved caspase-3, Bcl2, and PSD-95 in N2A cells was confirmed by immunocytochemistry. Cells in all experimental groups were washed three times with PBS, fixed with 4% paraformaldehyde for 3 h, and then washed with PBS. N2A cells were permeabilized with 0.025% Triton X-100 and blocked for 1 h with dilution buffer (Invitrogen, Carlsbad, CA, USA). The following primary antibodies: anti-rabbit BDNF (1 : 500, Abcam, Cambridge, MA, USA), anti-rabbit cleaved caspase-3 (1 : 500, Santa Cruz, Santa Cruz, CA, USA), anti-rabbit PSD-95 (1 : 500, Millipore, Massachusetts, MA, USA), anti-mouse Bcl2 (1 : 500, Millipore, Massachusetts, MA, USA) were prepared in dilution buffer, added to samples, and incubated for 3 h. Primary antibody was then removed and cells were washed three times for 3 min each with PBS. Later, samples were incubated with FITC-conjugated goat, anti-rabbit (1 : 200, Jackson Immunoresearch, PA, USA), or rhodamine-conjugated donkey, anti-mouse secondary antibodies (1 : 500, Millipore, Massachusetts, MA, USA) for 2 h. Cells were washed again three times for 3 min each with PBS and stained with 1 μg/mL DAPI (1 : 100, Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature. Fixed samples were imaged using a Zeiss LSM 700 confocal microscope (Carl Zeiss, Thornwood, NY, USA).

The mice were transcardially perfused with normal saline, followed by a precooled 4% paraformaldehyde solution. The brains were postfixed with 4% paraformaldehyde for 12 h and then transferred to a 30% sucrose solution. Coronal sections were cut at a thickness of 14 μm using a cryotome (Leica, CM1950, German). The sections were first washed three times with PBS for 10 min each and then permeabilized with immunostaining permeabilization buffer containing Triton X-100 (P0096, Beyotime, China) for 15 min, followed by 15 min in immunostaining blocking buffer (P0260, Beyotime, China). The sections were then incubated with primary antibody (rabbit anti-cFOS, 1:500, CST, USA) at 4°C for 72 h. After three 10-min washes in PBS, the sections were incubated with the corresponding secondary antibody (FITC-conjugated goat anti-rabbit; 1:400, Jackson Immunoresearch, USA) for 1 h at room temperature, followed by three 10-min washes in PBS before slide mounting. Immunofluorescence images were captured using laser scanning confocal microscopy (Olympus, FV1000, Japan) and then analyzed by a blinded researcher using Fiji software.

Cells were grown on a glass coverslip, fixed and permeabilized with methanol (−20 °C), and washed with PBS before adding appropriate primary and secondary antibodies. Cells were stained with the following primary antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-α-tubulin (Sigma-Aldrich), mouse anti-γ-tubulin (Sigma, Germany), rabbit anti-CEP250 (Proteintech, Rosemont, IL, USA), rabbit anti-Rootletin (Santa Cruz Biotech), rabbit anti-SIK2 (Cell signaling), mouse anti-SIK2 (Biolegend, San Diego, CA, USA), and rabbit anti-EB1 (Santa Cruz Biotech). The secondary antibodies: FITC-conjugated donkey anti-mouse, FITC-conjugated goat anti-rabbit, or Cy3 conjugated goat anti-mouse (Jackson Immunoresearch, West Grove, PA, USA). DNA was stained using DAPI (4′,6-diamidino-2-phenylindol-dihydrochloride) (Roche, Mannheim, Germany). Slides were examined using an Axio Imager 7.1 microscope (Zeiss, Göttingen, Germany), and images were taken using an Axio Cam MRm camera (Zeiss, Göttingen, Germany).