Fluorescein conjugated secondary antibody

Cells were treated with AlexaFluor594-labelled α-synuclein fibrils before being washed 3 times in PBS and fixed with 4% paraformaldehyde at different time-points. Cells were made permeable with 0.25% Triton-X100, blocked with 10% horse serum (Vector Labs, Burlingame, CA, USA), stained with anti-heparan sulfate antibody 10E4 (AMSBIO) at a dilution of 1:100, followed by staining with fluorescein-conjugated secondary antibody (1:100, Vector Labs). Slides were mounted with ProLong® Gold Antifade Mountant (Thermo Fisher Scientific) and studied with a Axiovert 35 microscope (Zeiss, Germany) with an attached MRC1024 laser scanning confocal microscope system (BioRad, Hercules, CA, USA) and analyzed with Image J v1.43 software (NIH, Bethesda, MD, USA).

RPE monolayers grown on Transwell filters [31 (link)]) were fixed in 4% PFA, and subsequently permeabilized with 0.1% Triton-X 100 for 15 min. Samples were blocked in 5% normal goat serum followed by incubating with either OGC (1:100) and DIC (1:100) rabbit polyclonal antibodies overnight at 4 °C. The cells were washed and incubated with fluorescein conjugated secondary antibody (Vector Labs, Burlingame, CA, USA) for 30 min at room temperature. Transwell membranes were cut and removed from the inserts with a fine razor and mounted on a microslide. The specimen was viewed on an LSM 770 laser-scanning microscope and (Carl Zeiss, Thornwood, NY, USA).