Immunocytochemistry

Fixed cells or frozen tissue sections were blocked and permeabilized with PBS containing 4% bovine serum albumin, 2% goat serum, 100 mM glycine and 0.3% Triton X-100. Primary antibody was applied overnight at 4 °C diluted in blocking solution, washed 3 times in PBS, incubated for 2 h in the secondary antibody at room temperature, with DAPI, then washed four times in PBS, mounted in Vectashield® and imaged with confocal microscope Zeiss LSM800 or imaged with conventional microscope Zeiss Axiovert 135 M equipped with the camera Leica DC 350 FX CCD monochrome. The following antibodies were used: anti-acetylated tubulin (1/400, clone 6-11B-1, Sigma), anti p62 (1/200, GP62-C, Progen, Germany), anti-Ubiquitin conjugated protein1 (1/100, BML-PW8810, Enzo), anti - Lc3b (1/100, #2775, Cell Signaling Technology, Danvers), anti-beta3 tubulin (1/500, TUJ1, Biolengend), anti-NEFM (1/500, Poly28410, Biolegend), anti-NEFH (1/2000, Smi32, Biolegend), anti-cleaved caspase 3 (1/100; 5A1E, Cell Signaling Technology, Danvers).