Visualization of Myelinated Pyramidal Neurons

Human pyramidal cells were filled with 5 mg/mL biocytin and fixed in 4% PFA overnight. Slices were washed with PBS and stained with streptavidin-Cy3 secondary antibody (1:300, Invitrogen) in a PBS-based solution containing 0.4% Triton X-100 and 5% bovine serum albumin (BSA; Sigma-Aldrich, The Netherlands) during 3 h at room temperature. Slices were mounted and coverslipped. Slices were recut in 40 μm slices and then blocked in PBS containing 0.4% Triton X-100 and 5% BSA and posteriorly stained using mouse anti-MBP (1:300, Santa Cruz, F-6, sc-271524) in PBS, 0.4% Triton X-100 and 5% BSA during 72 h at 4°C. Secondary antibodies Alexa-488 anti-mouse and streptavidin-Cy3 were diluted in PBS, 0.4% Triton X-100 and 5% BSA during 3 h at room temperature. Slices were then cover-slipped and sealed for imaging.