Immunofluorescencent Staining of A-synuclein, TFEB, GCase and LAMP1 in treated cultured cells

This protocol can be used to detect the amount and localization of endogenous a-synuclein, TFEB, GCase and LAMP1 by confocal immunofluorescence. In our hands, we performed the same protocol in undifferentiated SH-SHY5Y parental cell line and in SH-SY5Y expressing a mutant form of the SNCA gene which leads to a rapid aggregation of a-synuclein protein following its interaction with lipid-membrane organelles. In this mutant model, the familiar PD mutation E46K was “amplified” by introducing two analogous extra-lysine mutations into the nearby KTKEGV repetitive motive in positions E35K, E46K, and E61K. Subsequently, we applied a differentiation protocol both to the SH-SY5Y parental cell line and the 3K-SNCA overexpressing ones. In differentiated cells, the same immunostaining protocol was again conducted.