Microtubule Dynamics Modulation by 2-AAPA

TE-13 cells were incubated with 2-AAPA at 37°C in a humidified atmosphere of 5% CO₂ for 4 h. The immunofluorescence staining of microtubules were processed as described earlier [25 (link)]. Briefly, the TE-13 cells were fixed in 4% paraformaldehyde at room temperature for 1 h. The cells were washed 3 times with PBS and incubated with cell permeable solution (0.1% Na-citrate, 0.1% Triton-X-100 in PBS) at room temperature for 1 h. After incubation with the blocking solution (5% bovine serum albumin in PBS) overnight at 4°C, microtubules were visualized by incubating with a mouse monoclonal anti-α-tubulin-FITC (1:200) at 37°C for 2 h, and nuclei were stained with DAPI (1 μg/mL). Fluorescent images were taken with an Olympus Fluoview FV1200 microscope. Paclitaxel and vincristine sulfate were employed as positive controls for microtubule stabilization and depolymerization, respectively.