Dmi6000cs confocal microscope

Cells were seeded on coverslips coated with 1% gelatin, fixed with 4 % PFA for 10 min on ice followed by 20 min at 22°C and quenched with 50 mM ammonium chloride for 30 min at 22°C. The coverslips were then permeabilized with 0.2% Triton X-100 for 30 min at 22°C and blocked with a solution containing 1% BSA, with added 0.5% saponin, 0.1% Triton X-100 for additional permeabilization for 10 min at 22°C. Cells were then incubated with anti-α-galactosidase A (Sigma, Germany) for 1 h at 22°C. Horseradish peroxidase–conjugated goat anti-rabbit was used as a secondary antibody to visualize the GAA protein. Evaluation of slides was done using a Leica DMI6000CS confocal microscope with a HCXPLAPO 40× 0.75–1.25 oil objective. The gain setting remained constant throughout.

IF assay for tissue samples was performed according to a previous protocol (Gao and Chen, 2013 (link)). The samples were incubated with rabbit anti-BAF45D (1:100, Proteintech, Chicago, IL, USA), mouse anti-GFAP (1:100, Proteintech, Chicago, IL, USA), mouse anti-NEUN (1:100, Millipore, Belecula, CA, USA) and mouse anti-beta-III-tubulin (1:200, Millipore, Belecula, CA, USA) overnight at 4°C. After washed with PBS, the samples were incubated with Alexa Flour-488 anti-mouse (1:500) and Alexa Fluor-594 anti-rabbit (1:500) antibodies. The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI). A NIKON Eclipse 80i fluorescence microscope and a NIKON Eclipse Ti-S inverted fluorescence microscope were used for visualization. In some cases, a Leica DMI6000CS confocal microscope was used for the visualization. More descriptions of the antibodies used for IF were shown in Table S1.

Multi-label immunofluorescent staining with two or three rabbit antibodies was carried out using the tyramide signal amplification (TSA) kit (PerkinElmer) according to the manufacturers’ instructions. This allows for a very extensive dilution of the primary antibody, reducing cross-reactivity of the second secondary antibody to the first primary antibody to a minimum. Double-labeling was carried out with one TSA-reaction followed by normal immunofluorescent staining. Triple-labeling entailed two TSA-reactions followed by normal immunofluorescent staining. Secondary antibodies for TSA-reactions were swine anti-rabbit- HRP (Dako). For immunofluorescence donkey anti-rabbit conjugated to FITC or Cy3 (Jackson ImmunoResearch) or goat anti-rabbit Alexa Fluor 594 (Invitrogen) were used. Images were captured using a Leica DMI6000 CS confocal microscope connected to a Leica TCS SP5 scanner.