Anti tuj1 antibody

The following primary antibodies were used for immunohistochemistry experiments: primary Anti-SIK3 antibody (ab110987, Abcam, Cambridge, UK, and as a control: LS-c120369 SIK3 Lifespan Bioscience, Seattle, USA) at 1 : 50 (P0, P5) or 1 : 40 (4 weeks) concentration; anti-peripherin antibody (ab4666, Abcam, Cambridge, UK) at a 1 : 100 concentration; anti-TUJ1 antibody (Covance, New Jersey, US) at a 1 : 100 concentration; anti-GFAP antibody (ab53554, Abcam, Cambridge, UK) at a 1 : 50 concentration. The secondary antibody, biotin-conjugated donkey anti-rabbit (711-065-152), was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Images of the antibody stained sections were taken using a Zeiss Axioskop MOT light microscope. Image processing was performed in Adobe Photoshop CS5.

Five days after primary cortical cells were seeded, neurons in the microfluidic chamber were fixed by 4% paraformaldehyde, incubated with anti-Tuj-1 antibody (1:500, Covance Princeton, NJ) overnight and followed with Cy3 labeled secondary antibody. Nuclei were counter-stained by DAPI (1:10000, Thermo Fisher Scientific Inc). Axons were recognized by Tuj-1 positive fibers and quantified with ImageJ software 1.34 to show total axon length (μM) in a field. To rule out possible dendrite contamination in microgrooves, we selected cross-line between the ends of microgrooves and axonal compartment as the reference line and do binning through the entire axonal compartment. At least fifty randomly selected axon fields in more than 8 microfluidic chambers from 3 different experiments per group were quantified by experimenters blinded to each culture condition.

Immunofluorescence staining was performed according to a standard protocol. Anti-Tuj1 antibody was purchased from Covance. Anti-Gfap (ab4674) antibody was purchased from Abcam. Slides were mounted with mounting medium (Ibidi, GmbH) containing DAPI (Invitrogen).