Immunofluorescence Analysis of Cytoskeleton

Immunofluorescence analyses were performed to study the NT2D1 cytoskeleton changes analyzing vinculin and F-actin localization by using confocal microscopy analysis. The immunofluorescence protocol was reported in detail in [8 (link)]. Cells were fixed, permeabilized, and incubated overnight with anti-vinculin (Santa Cruz, cat. sc-73614; 1:50). After washings, cells were incubated with the FITC-conjugated donkey anti-mouse IgG secondary antibody (Jackson ImmunoResearch, cat. 715-095-150, dil. 1:200). TO-PRO3 iodide fluorescent dye 642/661 (1:5000 in PBS, Invitrogen, cat. T3605, Carlsbad, CA, USA) was used for nuclei staining. Rhodamine phalloidin (Invitrogen Molecular Probes Eugene 1:40 dilution) was used for F-actin detection. Samples were analyzed by using a Leica confocal microscope (laser scanning TCS SP2 equipped with Kr/Ar and He/Ne lasers, Mannheim, Germany).
The colocalization of vinculin and F-actin at the focal contacts was analyzed by Leica confocal software LAS-AF-Lite_2.6.3.