Neuronal class 3 β tubulin tuj1

Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, permeabilized, and stained in a blocking solution containing the antibodies, 0.03 g/ml bovine serum albumin (BSA, Sigma), 10% goat serum (Sigma), and 0.3% Triton X-100 (Sigma) in PBS for 2 hr. The samples were washed before incubation with secondary antibodies for 1h at RT. The samples were then mounted in Fluoro-Gel (Electron Microscopy Sciences, Hatfield, PA, USA) for fluorescent imaging.
Antibodies against the following proteins were applied for immunofluorescence (IF). Monoclonal antibodies: Neuronal class III β-tubulin (Tuj1) 1:500 (Covance, Princeton, NJ), MAP2 1:500 (BD Biosciences, Franklin Lakes, New Jersey). Rabbit polyclonal antibodies: Neuronal class III β-tubulin (Tuj1) 1:500 (Covance). Secondary antibodies used for IF were Alexa Fluor 594 goat anti-rabbit secondary antibody 1:200 (Life Technologies/Invitrogen), or Alexa Fluor 488 goat anti-mouse secondary antibody 1:200 (Life Technologies/Invitrogen) and cell nuclei were stained with 4,6-Diamidino-2-phenylindole (DAPI) 1:5000 (Life Technologies/Invitrogen).