Alexafluor 488 goat anti rabbit iggs

For microscopy analysis, BV2 cells were grown on coverslips for 24 hours and treated with the corresponding reagent for 2 h. Cells were fixed with 4% Paraformaldehyde for 15 min at room temperature, and blocked/permeabilized in HEPES 10 mM, 3% bovine serum albumin, 0.3% Triton X-100 diluted in PBS at room temperature for 1 h. Later, the slides were left overnight in a humidified chamber at 4 °C with the indicated primary antibody diluted on blocking solution. The day after, the samples were washed 3 times with 0.1 Tween/PBS for 5 min and incubated with the corresponding secondary antibody AlexaFluor® 488 Goat anti-Rabbit IgGs or AlexaFluor® 488 Goat anti-Mouse (1:500; Molecular probes/Invitrogen, Waltham, MA, USA) at room temperature for 1 h. Slides were mounted using ProLong Gold antifade mounting medium containing DAPI used to stain the nuclei. Samples were analyzed under Zeiss LSM700 confocal laser scanning microscopy equipped with ZEN Zeiss software. Antibodies are listed in the Supplementary Table 1.

For confocal microscopy analysis, the adherent mammalian cells were grown on coverslips for 24 h. 4% Paraformaldehyde-fixed cells were blocked in HEPES 10 mM, 3% bovine serum albumin (Sigma-Aldrich, A9418), 0.3% Triton X-100 (Sigma-Aldrich, T8787) diluted in PBS (1 h at room temperature) and incubated with the indicated primary (4°C, overnight) and secondary AlexaFluor® 488 goat anti-rabbit IgGs (1:1000; Molecular probes/Invitrogen, A11008; room temperature, 1 h) antibodies. Antibodies are listed in above section. Nuclei were counterstained with Hoechst 33,342 (Molecular Probes/Invitrogen, H3570). Samples were mounted with Vectashield® Antifade Mounting Medium (Vector Laboratories, H-1900) and analyzed with Zeiss LSM700 confocal laser scanning microscopy (Oberkochen, Germany).