Ab24640

Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 4% to 20% gradient gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred onto polyvinylidene fluoride or nitrocellulose membranes (Thermo Fisher Scientific). Membranes were blocked either with 5% bovine serum albumin (BSA, Sigma-Aldrich) or 5% milk in Tris-buffered saline (TBS). Proteins were analyzed with rabbit antibodies against myelin basic protein (Mbp, 1:400, AB980, Thermo Fisher Scientific), microtubule-associated protein 2 (Map2, 1:500, ab24640, Abcam, Cambridge, UK) and β-Tubulin (1:4000, ab6046, Abcam) in 5% milk (for Mbp) or 5% BSA (for Map2 and β-Tubulin) in TBS-Tween 20 (TBS-T, Sigma-Aldrich) overnight at 4 °C. Horseradish peroxidase-coupled donkey anti-rabbit (1:1000, GE Healthcare Life Science, Piscataway, NJ, USA) antibody was used as a secondary antibody and incubated with the membranes for 1 h at room temperature. Binding was detected using the chemiluminescent Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) on a Chemidoc XRS+ system (Bio-Rad). Pixel summation of individual bands was performed with ImageJ Software (NIH, Bethesda, MD, USA). The ratio between Mbp/Map2 and β-Tubulin was calculated for each animal.

Tissue samples for immunohistochemistry were collected after mice were perfused with PBS and 4% paraformaldehyde. Five millimetre thick tissue sections were deparaffinated in Histoclear and hydrated in a descending alcohol concentration series. Then, slides were incubated with TBS, and permeabilized with 0.2% Triton X-100 in TBS solution for 10 min and rinsed in TBS 0.025% Triton X-100. Blocking was performed with 10% foetal bovine serum, plus 1% BSA and 0.3 M glycine, in TBS, for 2 h at room temperature. Primary antibodies were always incubated overnight at 4°C, in 1% BSA in TBS. Secondary antibodies were incubated 1 h at room temperature. The slides were mounted in a fluorescent mounting medium (DAKO, Denmark) and imaging was performed on a laser scanning Confocal Microscope Leica SP5 AOBS SE, using the 40×/63× oil objective. In each set of experiments, the same batch of antibodies (primary and secondary) was used, and images were taken using the same settings, such as camera exposure times. Primary antibodies used were rabbit monoclonal C-terminal anti-Megalin (1:750, Abcam, ab24640) and anti-mouse recombinant TTR (1:250, custom made, Quantum Appligene, Illkirch, France). As secondary antibodies, Alexa Fluor 488 and 568 (1:750, Life Technologies) were employed.