Immunostaining of Cultured Hippocampal Neurons

Cultured hippocampal neurons were fixed in 4% PFA solution, permeabilized with 0.1% Triton X-100, blocked with 2% goat serum and immunostained as described previously^{38 (link)}. To improve the F-actin staining, phalloidin was incubated for 2h following the immunostaining steps with primary and secondary antibodies. Coverslips were mounted in Mowiol-DABCO for imaging. F-actin was stained with phalloidin-STAR635 (Abberior, cat. 2-0205-002-5, 1:50 dilution). Dendrites were identified using a staining against the microtubule-associated-protein-2 with Rabbit-anti-MAP2 PAB (Milipore Sigma, cat. AB5622, 1:1000) and GAR-STAR488 SAB (Abberior, cat. 2-0012-006-5, 1:250). Axons were identified using a staining against the phosphorylated neurofilaments (SMI31) stained with the PAB mouse-anti-SMI31 (Biolegend, cat. 801601, 1:250) and the SAB GAM-STAR580 (Abberior, cat. 2-0002-005-1, 1:250).