Fluoview 10 asw software

Tau K18 fibrils (NBP2-76793, NovusBio) were labeled with Alexa Fluor succinimidyl ester dye 647 (A37573, Thermo) and transformed into cells as reported [43 (link)]. Control or SMS siRNA are transfected into SVG p12 cells (CRL-8621, ATCC) on coverslips in 12-well plates as mentioned above. Three days after transfection, the sonicated Tau K18 fibrils were added into the medium. Two days later, the cells were fixed with 4% FA for 15 min, permeabilized with 0.4% Triton X-100 for 5 min and stained with p62 antibodies and DAPI. The cells were mounted on glass slides with VECTASHIELD Antifade Mounting Medium (Vector Laboratories) and kept at 4 °C until imaging on an Olympus IX81 confocal microscope. Images were processed with FluoView 10-ASW software (Olympus). Quantification of the fibril loci was performed using ImageJ program, as previously described [44 ]. Briefly, the fibril loci were segmented with interactive h-maxima watershed method and then the size and intensity of recognized loci were automatically measured with the particle analyzing tool.

Flight muscles were dissected in cold PBS and fixed in freshly made 4% formaldehyde for 15 minutes. After 10 minutes washing in PBS containing 0.4% (v/v) Triton X-100 (PBTX) 3 times, the muscles were incubated with ATP5α Abs diluted in 0.4% PBTX containing 5% goat serum overnight at 4°C. The muscles were then incubated at room temperature with conjugated secondary Abs for 2 hours, followed by staining with DAPI for 10 minutes. After washing, the muscles were mounted on glass slides with VECTASHIELD Antifade Mounting Medium (Vector Laboratories) and kept at 4°C until imaging. Specimens were imaged using an Olympus IX81 confocal microscope. Images were processed using FluoView 10-ASW software (Olympus) and analyzed using ImageJ software.