Alexa 647 goat anti rabbit

To determine localization, immunohistochemistry (IHC) was performed on paraffin-embedded ON sections (10 μm) using the same antibodies to GBA1 (1:200), GBA2 (1:200), ASAH1 (1:100), ASAH2 (1:100), and α-synuclein (1:500) mentioned previously. The sections were deparaffinized and washed with PBS, blocked with 0.2% BSA for 30 minutes, and subsequently incubated with primary antibodies overnight. Sections were then incubated for 1 hour at room temperature with Cy5-conjugated goat anti-rabbit (1:1000, Abcam, Inc.), Alexa 647 goat anti-rabbit (1:1000, Abcam, Inc.), or Alexa 594-conjugated donkey anti-sheep (1:1000, Abcam, Inc.). Control samples (without primary antibody–treated sections) were examined side by side on the same slide. Fluorescence images were obtained with a laser-scanning confocal imaging system (Zeiss LSM510; Peabody, MA, USA).

All reagents used were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands) unless specified otherwise. The primary antibodies and probes used for monolayer assessment were Phalloidin-AF488 (Invitrogen, Carlsbad, CA, USA) diluted 1:100, Mouse anti-α-tubulin (#EP1332Y; Invitrogen, Carlsbad, CA, USA) diluted 1:500 and rabbit anti-Na+/K+ATP-ase (a kind gift from Dr. Jan Koenderink, Radboudumc, The Netherlands) diluted 1:500. The primary antibodies used for autophagy and vesicle trafficking assessment were rabbit anti-LC3 (Novus Biologicals, Abingdon, UK #NB600-1384SS) diluted 1:1000, mouse anti-P62 (SQSTM1) (#610832; BD Biosciences, Mississauga, ON, Canada) diluted 1:1000, mouse anti-LAMP1 (#sc-18821; Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:200, and rabbit anti-mTOR (#2983; Cell Signaling Technology, Leiden, The Netherlands) diluted 1:400. The secondary antibodies used for detection were Polyclonal goat anti-rabbit (#P0448, Dako products, Carpinteria, CA, USA) diluted 1:5000, and polyclonal goat anti-mouse (#P0447, Dako products, Carpinteria, CA, USA) diluted 1:5000. Additionally, Alexa-488 goat anti- mouse (#ab150113; diluted 1:500), Alexa-647 goat anti-rabbit (#ab150083; diluted 1:200), donkey anti-rabbit (#AF647; diluted 1:300), and donkey anti-mouse (#AF568; diluted 1:200) secondary antibodies were all from Abcam (Amsterdam, The Netherlands).