Fluorophore conjugated secondary antibodies derived from goat or donkey

HEK293T or Hela cells were seeded on fibronectin-coated glass coverslips and allowed to attach overnight. On the following day, cells were subjected to either amino acid starvation and stimulation with or without drug incubation and fixed in 4% paraformaldehyde (PFA) for 15 minutes at room temperature. The coverslips were rinsed twice with PBS and cells were permeabilized with 0.1% (w/v) saponin in PBS for 10 minutes. After rinsing twice with PBS, the slides were incubated with primary antibodies in 5% normal donkey serum (Jackson ImmunoResearch, 017-000-121) for 1 hour at room temperature, rinsed four times with PBS, and fluorophore conjugated secondary antibodies derived from goat or donkey (Life Technologies, diluted 1:400 in 5% normal donkey serum) were incubated for 1 hour at room temperature in the dark. The coverslips were then washed four times with PBS, mounted on glass slides using Vectashield (Vector Laboratories) and imaged on a spinning disk confocal system built upon a Nikon Eclipse Ti microscope with Andor Zyla-4.5 sCMOS camera.

HEK-293T or HEK-293A cells were seeded on fibronectin-coated glass coverslips in 6-well plates (35 mm diameter/well), at 300,000–500,000 cells/well and allowed to attach overnight. On the following day, cells were subjected to sterol depletion and restimulation, where indicated, and fixed in 4% paraformaldehyde (PFA) for 15 minutes at room temperature. The coverslips were rinsed twice with PBS and cells were permeabilized with 0.1% (w/v) saponin in PBS for 10 minutes. After rinsing twice with PBS, the slides were incubated with primary antibodies in 5% normal donkey serum (Jackson ImmunoResearch, 017–000-121) for 1 hour at room temperature, rinsed four times with PBS, and incubated with fluorophore-conjugated secondary antibodies derived from goat or donkey (Life Technologies, diluted 1:400 in 5% normal donkey serum, PBS) for 45 minutes at room temperature in the dark. After washing four times with PBS and one time with deionized water, the coverslips were mounted on glass slides using Fluoromount-G (SouthernBiotech, 0100–01) and imaged on a spinning disk confocal system built upon a Nikon Eclipse Ti microscope with Andor Zyla-4.5 sCMOS camera.