Elyra s1 super resolution microscope

Immunofluorescent staining was conducted to evaluate the formation and localization of Zera^®-induced protein bodies. Four-well chamber slides (AEC Amersham) were pre-coated with poly-L-Lysine (Sigma-Aldrich) and seeded with 30000 HeLa cells per well. Three days post transfection with pMExT gp140-Zera^®, pMEx Zera^®-gp140, pMExT gp140 or pMEx Zera^®-eGFP, cells were fixed with 4% paraformaldehyde for 10 min, washed with 1x phosphate buffered saline (PBS), permeabilized and blocked with 2% BSA-PBS supplemented with 0.25% Triton X-100 for 1 h at room temperature. Cells were double-stained overnight at room temperature with goat anti-HIV-1 gp120 and rabbit polyclonal antibodies to calnexin (Abcam), diluted 1:500 and 1:200, respectively in 2% BSA-PBS. Following washes with PBS, cells were incubated in the dark for 1.5 h with the fluorophore-conjugated secondary antibodies (1:500 donkey anti-Goat-Cy3 and 1:500 donkey anti-rabbit-Alexa488/Cy3) diluted in 2% BSA-PBS. The cells were then washed with PBS and incubated for 10 min with 1:5000 Hoechst nuclei stain diluted in PBS. Following washes in PBS, slides were mounted with antifade-moviol mounting media. Slides were imaged with a confocal microscope (Carl Zeiss 880 LSM confocal with Fast Airyscan technology and the Elyra S1 super-resolution microscope).