Abn78

Immunofluorescence was performed on coronal 14 μM cryosections of young adult (3–4 weeks old) mouse main olfactory epithelium. Slides were dried for 10 min at room temperature, fixed in 4% paraformaldehyde (PFA), phosphate-buffered saline (PBS) 1×, pH7.4, and washed three times for 5 min in PBST (PBS 1×, 0.1% Triton X-100). Sections were blocked for 1 h in blocking buffer (PBS 1×, 4% donkey serum (Sigma), 1% Triton X-100). The following primary antibodies were used at the specified dilution: mouse α-Rbfox1 (Millipore 1D10, 1:100), rabbit α-Rbfox2 (Bethyl Laboratories, A300-864A, 1:1000), rabbit α-Rbfox3 (Millipore ABN78, 1:200), and goat α-Calmegin (Santa Cruz N-16, 1:50). Primary antibodies were diluted in blocking buffer, and sections were incubated overnight at +4 °C. Slides were washed three times for 5 min in PBST and then incubated for 1 h at room temperature with blocking buffer containing secondary antibodies (Jackson Immunoresearch) (1:500) and DAPI (4',6-diamidino-2-phenylindole; 1:1000). Slides were washed three times for 5 min in PBST and mounted with Vectashield (Vector Laboratories). Images were taken on a Zeiss LSM700.

After treatment, the animals previously anesthetized were perfused transcardially with 4% paraformaldehyde solution, and brains were processed as previously described^{52 (link)}. Sections were then incubated with anti-sigma 1 receptor rabbit (Abcam ab53852) combined with anti-nestin mouse (Abcam ab6142), anti-BrdU mouse monoclonal (DAKO M0744) combined with anti-nestin rabbit (Abcam ab7659), anti-NeuN rabbit (Millipore ABN78), or anti-doublecortin (DCX, Santa Cruz sc-8066) antibodies at 4 °C overnight, washed three times and incubated with AlexaFluor 488 goat anti-mouse and Alexa-647 goat anti-rabbit secondary antibodies for 1 h at room temperature. After rinses, sections were mounted with Vectashield. Images were obtained using a LSM710 laser-scanning spectral confocal microscope (Zeiss). Confocal microscope settings were adjusted to produce the optimum signal-to-noise ratio. Five animals from each experimental group were analyzed.