Plan neofluar 40 1.3 oil lens

HEK293T cells on chambered glass slide were washed once with PBS and fixed for 15 min in 4% formaldehyde in PBS at 25°C. Cells were then washed once in PBS and permeabilized in 0.1% Triton X-100 in PBS for 3–5 min at 25°C. Permeabilized cells were washed three times for 5 min each in PBS, blocked for 60 minutes in blocking solution (1% BSA in 1x PBS) and then incubated at 4°C overnight with either rabbit anti-PDE6C [27 (link)] and mouse anti-HA antibodies (BioLegend), or mouse anti-FLAG (Sigma) diluted (1:2000) in the blocking solution. After washing in PBS three times for 5 min each, where indicated, cells were incubated in the dark for 1 hour in either Alexa Fluor 568-conjugated goat anti-rabbit or Alexa Fluor 488-conjugated goat anti-mouse (Life Technologies) or Alexa Fluor 647-conjugated F(ab′)2-Goat anti-mouse IgG (H+L) secondary antibody (Thermo Scientific) (1:2000) diluted in the blocking solution. Cells were washed three times for 5 min each in PBS, and the nuclei were counter stained with To-Pro-3 (1:1000) (ThermoFisher) for 30 min in the dark at 25°C. Cells were mounted using Vectashield mounting medium (Vector Laboratories, Inc) and imaged using Plan-Neofluar 40×/1.3 oil lens and a LSM 510 confocal microscope (Zeiss).

The silencing experiments were visualized using a UV 510 confocal microscope (Carl Zeiss, Oberkochen) and a Plan-Neofluar 40×/1.3 oil lens. MDA-MB-231 eGFP cells were plated in glass bottom dishes (Ibidi, Madison), transfected as described above. 3 days upon transfection, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 20 min at RT for imaging. For GFP imaging, the 488 nm line of an argon laser was used as excitation, and emission was collected using a BP 505–550 filter. The uptake experiments were visualized using a LSM 710 confocal microscope (Carl Zeiss, Oberkochen) and a Plan-Apochromat 63×/1.40 oil lens. MDA-MB-231 cells were transfected with Alexa 456 fluorescently labeled nanoconstructs. The next day, cells were washed three times with PBS and fixed with 4% paraformaldehyde for 20 min at RT for imaging. For Alexa 546 imaging, a DPSS 561 laser was used for excitation, and emission was collected between 566 and 680 nm.