Na plan apochromat vc oil immersion objective

Transfected HEK293 cells on glass coverslips were rinsed 24 hours following transfection two times in prewarmed phosphate buffered saline (PBS), fresh growth media supplemented with 50 μM biotin was added and the cells incubated overnight at 37°C. The following day, the cells were rinsed two times in PBS, fixed in 4% paraformaldehyde in PBS and permeabilized in 0.25% Triton X-100/PBS for 5 minutes. Cells were then rinsed in PBS and blocked in immunoblock buffer (2% goat serum, 3% bovine serum albumin, 1X PBS) before the addition of antibodies (1:400 mouse anti-HA (Thermo Fisher Scientific) or Streptavidin-HRP (Genscript, Piscataway, NJ, USA)) and incubated overnight at 4°C. Secondary antibody was used to visualize the HA epitope (1:50 goat anti-mouse Alexa 594 (Thermo Fisher Scientific)). To amplify the Streptavidin-HRP signal, tyramide signal amplification was used (TSA-FITC) according to manufacturer’s instructions (PerkinElmer). DAPI (Thermo Fisher Scientific) was added to all coverslips to visualize cell nuclei. Coverslips were rinsed and mounted in Fluoromount G (Electron Microscopy Science, Hatfield, PA, USA) and imaged on a Nikon TiE inverted epifluorescence microscope equipped with a 100x/1.4NA Plan-Apochromat VC oil immersion objective, a Lumencor SOLA LED light source for epifluorescence illumination, and an Andor iXon Ultra 897 EMCCD camera.