Fitc conjugated donkey anti mouse igg secondary antibody

As we previously described^{31 (link)}, the cells were fixed in chilled acetone and methanol (1:1) for 20 min at −20 °C and permeabilized with 0.1% Triton X-100. Mouse anti-influenza NP primary antibody were incubated 37 °C for 1 h and followed by FITC-conjugated donkey anti-mouse IgG secondary antibody (Jackson ImmunoResearch, PA, USA). After washing with PBS the slides were mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories). The stained cells were examined under fluorescence microscope Nikon80i imaging system.

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.