Mab326

Mice were sacrificed by decapitation and the brain rapidly removed, hippocampi promptly dissected and homogenized as described [25] (link). Proteins were denatured, reduced and separated on 10% Tris-Glycine SDS-PAGE gels (Anamed, Germany). After transfer, nitrocellulose membranes were probed with primary antibodies specific for protein tau and tubulin as described [25] (link) and for CNPase (Millipore - MAB326) as marker for myelin.

Isolated tissues were fixed in fresh 4% paraformaldehyde, embedded in paraffin, and cut into 5-6 µm sections using a Leica RM2155 microtome and Super Plus charged slides. Cerebellum (CB) slices were sectioned into sagittal sections, and hippocampus (HC) sections were in coronal orientation. For IHC, the primary antibody was used at optimal ratio in Antibody Diluent Reagent Solution (Life Technologies) and incubated overnight at 4°. Slides were then washed and incubated with secondary antibody (1:200) in antibody diluent for 1 hr at room temperature. Slides were further washed and stained with Hoecsht 3342 and scanned under Zeiss LSM 710 Confocal Microscope for further analysis. Primary antibodies: MAP2 (Abcam ab5392, 1:300), AUTS2 (Sigma HPA000390, 1:200), WBSCR17 (LSBio LS-B14501, 1:250), Calbindin D-28K (Sigma C9848, 1:300), Calretinin (Santa Cruz sc-365989, 1:300), CNPase (Millipore MAB326, 1:250), DRD2 (Millipore AB5084P, 1:200), GFAP (Invitrogen 180063, 1:300), GAD 65/67 (Santa Cruz sc-365180, 1:200), Tbr2 (Abcam ab23345, 1:100), cFos (Abcam ab190298, 1:200); Secondary antibodies (Thermo-Fisher Scientific): Goat anti-Mouse IgG (Alexa Fluor 488, A11001), Goat anti-Rabbit IgG (Alexa Fluor 594, A11012), Donkey anti-Rat IgG (Alexa Fluor 594, A21209).

Mice were euthanized and perfused with 15 ml ice-cold phosphate buffer saline (PBS), followed by 20 ml 4% ice-cold paraformaldehyde in PBS. Nerves and brains were harvested; fixed in 4% ice-cold paraformaldehyde overnight; cryoprotected with 10, 20, and 30% ice-cold sucrose in PBS for 4 h, 4 h, and overnight, respectively; and frozen in OCT (Sakura Inc). Ten Micrometre nerve sections and 40 μm brain sections were made for staining. Sections were blocked with 5% normal donkey serum plus 0.3% Triton® X-100 in PBS at room temperature for 1 h, incubated with primary antibody in block at 4 °C overnight. Antibodies and dilutions were: rabbit anti-p75NTR (Cell Signaling Technology, 8238, 1:2000), rat anti-MBP (Biorad, MCA409S, 1:100), goat anti-Gfap (Abcam, ab53554, 1:1000), mouse anti-NeuN (Millipore, mab377, 1:200), goat anti-Iba1 (Abcam, ab5076, 1:200), and mouse anti-CNPase (Millipore, mab326, 1:200). Following incubation with primary antibody, sections were washed three times with PBS, incubated with Alexa fluorophore-conjugated secondary antibodies (1:500, Invitrogen) in block at room temperature for 1 h, washed two times with PBS, incubated with 300 nM DAPI (Sigma) at room temperature for 10 m, washed two times with PBS, and mounted for confocal imaging (Perkin Elmer). TdTomato was strong endogenously and did not require an anti-RFP antibody.