Axioimager z2 motorized upright microscope

Cells were fixed at RT in 3.8% (vol/vol) formaldehyde in PBS and permeabilized in PBS containing 0.1% (vol/vol) saponin (Sigma-Aldrich) or 0.1% (vol/vol) Triton X-100 (Sigma-Aldrich). Coverslips were labeled with primary and secondary antibodies as previously described (Connell et al., 2009 (link)). In certain cases, soluble cytosolic proteins were removed to allow better imaging of membrane-associated protein, by prefixation treatment with a saponin-based cytosol extraction buffer, as previously described (Edwards et al., 2009 (link)). Slides were analyzed with an LSM880 confocal microscope (100× NA 1.40 oil immersion objective, 37°C), LSM710 confocal microscope (100× NA 1.40 oil immersion objective, 37°C), LSM780 confocal microscope (63× NA 1.40 oil immersion objective, 37°C), or AxioImager Z2 Motorized Upright Microscope (63× NA 1.40 oil immersion objective, RT, Axiocam 506; ZEISS), all with ZEN analysis software (ZEISS). Airyscan imaging was performed using an LSM880 microscope fitted with an Airyscan module, using Plan-Apochromat 63×/1.4-NA Oil differential interference contrast (DIC) M27 objective at RT. Images were subsequently processed using ImageJ, Adobe Photoshop, Adobe Illustrator, and ZEN analysis software. 3D image preparation was performed using Imaris (Bitplane). Colocalization of proteins was quantified using Volocity (PerkinElmer).

Cells were fixed at room temperature in 3.8% (v/v) formaldehyde in phosphate-buffered saline (PBS) and permeabilized in PBS containing 0.1% (v/v) saponin (Sigma) or 0.1% (v/v) Triton™ X-100 (Sigma). Coverslips were labelled with primary and secondary antibodies as previously described (Connell et al., 2009 (link)). Slides were analysed with a Zeiss LSM880 confocal microscope (100× NA 1.40 oil immersion objective, 37°C), or Zeiss AxioImager Z2 Motorized Upright Microscope (63× NA 1.40 oil immersion objective, room temperature, Zeiss Axiocam 506). Co-localization of immunofluorescence signals was quantified using Volocity Image Analysis Software. Images were subsequently processed using Adobe Photoshop and Illustrator and ZEN analysis software.

Cells were fixed at room temperature (RT) in 3.8% (vol/vol) formaldehyde in phosphate buffered saline (PBS) and permeabilized in PBS containing 0.1% (vol/vol) saponin (Sigma-Aldrich) or 0.1% (vol/vol) Triton X-100 (Sigma-Aldrich). Coverslips were labeled with primary and secondary antibodies as previously described (Connell et al., 2009 (link)). Slides were analyzed with an LSM880 confocal microscope (100 × NA 1.40 oil immersion objective, 37°C), LSM780 confocal microscope (63 × NA 1.40 oil immersion objective, 37°C), or AxioImager Z2 Motorized Upright Microscope (63 × NA 1.40 oil immersion objective, RT, Axiocam 506; ZEISS), all with ZEN analysis software (ZEISS). Images were subsequently processed using ImageJ, Adobe Photoshop, Adobe Illustrator, and ZEN analysis software. Co-localization of proteins was quantified using Volocity (PerkinElmer).