Spinning disk axio observer z1 confocal microscope

FM dye uptake experiments were performed using established protocol with modifications (Dong et al., 2015 ; Hoopmann et al., 2012 ). Cells were cultured in glass bottom tissue culture plates (µ–Dish, Ibidi). Fresh media was added to the cells along with 10µM FM1-43 or FM4-64 dye (Thermo Fisher). Cells were incubated for 5 mins at 37°C to allow FM dye internalization. Cells were gently washed using 1x PBS containing 0.5mM EGTA and were incubated in 1x PBS containing 1 mM ADVASEP-7 (Sigma) for 5 min to remove excess dye. Images were taken using an Olympus FV1000 confocal microscope with a 60x/1.42 oil objective or a Zeiss spinning disk Axio Observer Z1 confocal microscope with a 40x water Objective. FM1-43 was excited with a 488 nm laser and FM4-64 was excited with a 560 nm laser. All images were analyzed using ImageJ. The number of total cells in each image was counted, along with the number of cells that contained FM1-43/FM4-64 fluorescence puncta, to calculate the percentage of cells with dye internalization. Student’s t-test was used for statistical analyses.

Immunofluorescence was carried out using a published protocol (Conacci-Sorrell et al., 2014 (link)). Briefly, cells were cultured on glass coverslips or chamber slides, washed with PBS, and fixed with 4% paraformaldehyde for 30 mins. Next, cells were permeabilized using PBS buffer with 0.5% Triton X-100 for 5 min followed by treating with a blocking solution (Image IT FX, Invitrogen) for 30min. Cells were incubated with primary antibodies (1:100) for 1 hour. At the end of incubation, cells were washed 5x with 1x PBS and incubated with secondary antibodies for 1 hour. Samples were mounted to a microscope slide using ProLong Diamond Antifade Mountant (Thermo Fisher). All images were collected using an Olympus FV1000 microscope with a 60x/1.42 oil objective. To estimate the amount of membrane-bound and cytosolic mCherry-tagged EndoA3 variants, fluorescence images were collected using a Zeiss spinning disk Axio Observer Z1 confocal microscope with a 60x water objective, and were analyzed using ImageJ (Boucrot et al., 2015 (link)). Background fluorescence intensity was corrected for each image.