2.7. Immunofluorescence studies on cells

Cells on coverslips were fixed in 4% PFA solution in PBS for 15 minutes at room temperature and then washed with PBS. Cells were blocked with 5% bovine serum albumin and 0.3% Triton X‐100 in PBS at room temperature for 60 minutes. The coverslips were incubated with anti‐β3 tubulin antibodies (Cell Signaling Technology, Danvers, Massachusetts; CS5568; 1:500 dilution) overnight at 4°C and then with secondary antibodies for 1 hour at room temperature. Cell nuclei were visualized with DAPI. Slides were mounted with ProLong Gold Antifade Reagent and imaged using a TCS SP5 II confocal microscope. The confocal images were loaded into ImageJ with NeurphologyJ HT plugin. The number of neurites and total neurite length in the images were calculated using “NeurphologyJ HT” as described.⁴⁶