Cy5 conjugated 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).

To study integrin regulation of MMP expression, primary mouse anti-human integrin β1 (clone P4C10), integrin β2 (clone MEM48), integrin β3 (clone B3A), FITC-conjugated anti-human integrin β1, and integrin αV (clone 272-17E6) Abs were used (all from Millipore, Hertfordshire, U.K.). FITC-conjugated goat anti-mouse IgG1 (Sigma-Aldrich, Dorset, U.K.), and Cy5 conjugated goat anti-rabbit (Abcam, Cambridge, U.K.) were used as secondary Abs. Mouse IgG1 and FITC-conjugated mouse IgG1 were the isotype controls (BD Diagnostics, Oxford, U.K.).