Rabbit β tubulin

Immunofluorescence analysis on SH‐SY5Y cells was performed as previously described 54. Briefly, after fixation with 4% Paraformaldehyde (PFA), cells were permeabilized in 0.2% Triton X100 and blocked by incubation with 10% normal goat serum (NGS, Gibco, Invitrogen, Milan, Italy) for 1 hr at room temperature (RT). The primary incubation was performed, overnight at 4°C, with the following antibodies: rabbit anti‐p75 (1:500, Chemicon Int. Inc., USA), mouse anti‐S‐100 (1:100; Sigma‐Aldrich, Saint Louis, Missouri, USA), rabbit anti‐HIF1A (1:200; Sigma‐Aldrich, Saint Louis, Missouri, USA), mouse anti‐Cx43 (1:150, Cell Signaling, Danvers, Massachusetts, USA) and rabbit β‐tubulin (1:200, Cell Signaling, Danvers, Massachusetts, USA).
After washing, slides were incubated with the appropriate secondary antibodies: fluorescence isothiocyanate (FITC) labelled anti‐rabbit antibody (1:200, Chemicon, Int. Inc., USA) and Cy3 labelled antimouse antibody (1:1000 Chemicon, Int. Inc., USA) for 1 hr at RT. Nuclei were stained with DAPI (1:1000) for 5 min. Finally, slides were mounted in fluorescent mounting medium Permafluor (Thermo Scientific, Wilmington, USA) and digital images were acquired using a Leica DM IRB fluorescence microscope and with Leica TCS SP8 confocal microscope. Nonspecific staining of cells was observed in control incubations in which the primary antibodies were omitted.