Immunostaining of Cryosectioned Samples

The 18-µm thick cryosections were immunostained following previously described procedure^{41 (link)}. Briefly, upon drying sections were incubated in the blocking solution (PBS + 0.25% Triton + 10% goat serum) for 1 h at RT. Next primary antibody diluted in PBS + 0.25% Triton + 10% goat serum was added and sections incubated over night at 4 °C. After four washes with washing buffer (PBS + 0.25% Triton), secondary antibody diluted in PBS + 0.25% Triton was added and slides incubated for 2 h at room temperature. Slides were next washed four times with washing buffer, followed by two short washes with ddH2O. Primary antibodies: phosphorylated Vimentin (Ser55) (D076-3S, MBL International), TUJ1 (MMS-435P, Covance) and HUWE1 (A300-486A, Bethyl Laboratories). Secondary antibodies: Goat anti-Rabbit IgG (H + L) Secondary Antibody, Alexa Fluor® 568 conjugate (A-11011, Thermo Fischer) and Goat anti-Mouse IgG (H + L) Secondary Antibody, Alexa Fluor® 488 conjugate (A-11029, Thermo Fischer). Microscopy was carried out using a Leica SP8 confocal microscope equipped with ×40 oil immersion lens, using Huygens software.