Cerebellar cortex samples from the same subjects served as controls. Brain tissue was frozen and stored at −80°C until further analysis. Tissue samples were thawed and Dounce homogenized in 10 ml lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM magnesium

acetate, 0.1 mM EDTA, 10 mM Tris-HCl [pH 8.0], 0.1% Triton X-100, and 1 mM DTT). Homogenized samples were suspended in 20 ml of sucrose solution (1.7 M sucrose, 3 mM magnesium acetate, 1 mM DTT, and 10 mM Tris-HCl [pH 8.0]), layered onto a cushion of 10 ml sucrose solution, and centrifuged at 36,500 × g for 2.4 hr at 4°C. The isolated nuclei were resuspended in nuclei storage buffer (NSB) (10 mM Tris [pH 7.2], 2 mM MgCl2, 70 mM KCl, and 15% sucrose) AZD2281 solubility dmso for consecutive immunostaining and flow cytometry analysis. Isolated nuclei were stained with mouse NeuN (A-60) (Millipore, 1:1,000), rabbit Fox3 (Atlas Antibody, 1:300), mouse HuD (E-1) (Santa Cruz, 1:100), mouse HuD/HuC 16A11-biotin (Invitrogen, 1:300), or goat Sox10 (R&D, 1:300). NeuN (A-60) antibody was directly conjugated to Alexa 647 (Invitrogen Antibody Labeling Kit Alexa 647). All other primary antibodies were visualized with appropriate secondary antibodies conjugated to Alexa 488 (1:500), Ibrutinib ic50 Alexa 647

(1:500) (Invitrogen), or R-phycoerythrin (PE) (Santa Cruz, 1:100). Flow cytometry sorting was performed with a BD FACS Diva and flow cytometry analysis was performed with a BD FACS Aria instrument. Olfactory bulbs were fixed in 4% formaldehyde buffered in PBS for 24 hr and embedded in low-melting paraffin (52°C–54°C), according to standard

procedures. Olfactory bulbs were sectioned (5 μm) longitudinally and orthogonally according to their long axis. For immunohistochemistry, sections were deparaffinized in xylene and rehydrated in a descending ethanol series. Antigen retrieval was performed in citraconic acid solution (pH = 7.4; 0.05% citraconic acid) for 20 min in a domestic steamer (Namimatsu et al., 2005). The sections were allowed to cool down for 20 min before immunostaining was started. Sections were incubated with the respective primary antibody overnight at 4°C: mouse NeuN (Millipore A-60 clone; 1:100), rabbit Fox3 (Atlas Antibody, 1:300), goat Sox10 (R&D, 1:100), rabbit calbindin (Abcam, 1:200), chicken MAP-2 (Abcam, 1:1,000), rabbit calretinin (Abcam, found 1:200), rabbit parvalbumin (Abcam, 1:1,000), rabbit tyrosine hydroxylase (TH) (Millipore, 1:1,000), rabbit GAD65/67 (Millipore, 1:500), rabbit CNPase (Atlas Antibody, 1:400), mouse GFAP (Sigma Aldrich, 1:1,000), rabbit Iba1 (Wako, 1:1,000), mouse HuD (E-1) (Santa Cruz, 1:100), and mouse HuD/HuC 16A11-biotin (Invitrogen, 1:100), and visualized with the matching secondary antibody and streptavidin conjugated to Alexa 488, 546, or 647 (1:1,000, Invitrogen). All experiments were carried out in a clean room (ISO8) to prevent any carbon contamination of the samples. All glassware was prebaked at 450°C for 4 hr.