Immunocytochemical Analysis of Molecular Markers

Immunocytochemistry was performed as previously described [26 (link)]. Briefly, after fixing cells with 4% paraformaldehyde in phosphate buffered solution (PBS) (Cat. # AAJ19943K2, Thermo Fisher Scientific Inc.), they were permeabilized by adding cold methanol and then probed with the indicated primary antibodies. Antibodies used are as follows: anti-α-synuclein (#2642, 1:1000, Cell Signaling Technology), anti-phospho-α-synuclein (Ser129) (ab51253, 1:1000, Abcam plc.), anti-tyrosine hydroxylase (MAB318-AF488, 1:100, Millipore), anti-FOXA2 (ab108422, 1:300, Abcam plc), anti-phospho-IRS1 (Ser302) (Cat. # 2384, 1:1000, Cell Signaling Technology), and anti-IRS-1 (Cat. # 2382, 1:1000, Cell Signaling Technology). Fluorescence intensities from images of randomly selected microscopic fields of cells were semi-quantitatively analyzed based on staining positivity indices estimation using the NIH ImageJ software (https://imagej.nih.gov/ij/) as previously described by Jensen [27 (link)].