Ixon 897 emccd camera

For live imaging, cells were plated onto 35 mm glass bottom dishes (ibidi GmbH, Germany) and transiently transfected using Fugene HD (Promega, USA) 24 hr prior to imaging. All cultures were maintained at 37°C and 5% CO₂ for the duration of the experiment. Confocal microscopy was performed on a Zeiss 880 equipped with a Plan-Apochromat 63×/1.4 NA Oil DIC M27 objective and an Airyscan detector. TIRF imaging was performed using the Zeiss Elyra system fitted with a Plan-Apochromat 100×/1.46 NA Oil objective and an Andor iXon 897 EMCCD camera. In both cases, samples were illuminated using 488 nm, 561 nm or 633 nm lasers, and all data was collected using the Zen software (Zeiss).

Live imaging was performed on an N-SIM super-resolution system (Nikon, Japan) equipped with 100× Plan Apo TIRF lens (NA=1.49) and iXon 897 EM-CCD camera (effective pixel size 63 nm) (Andor, Ireland) in 3D-SIM mode (excitation laser line 488 nm, 120 nm Z-steps) under control of NIS-Elements 4.6 software. Raw image stacks (3 grating angles×5 phase shifts) were analyzed for image quality with the SIMcheck module of ImageJ software and processed using SIM module of NIS-Elements using parameters selected on the basis of Fourier transform analysis. Reconstructed stacks were further deconvolved using the Richardson-Lucy algorithm built into NIS-Elements.
Images for colocalization were processed using FIJI (Schindelin et al., 2012 (link); Schneider et al., 2012 (link)), including the Enhance Contrast feature to correct for bleaching and the Maximum Instensity projection feature for presentation in the paper.

STORM analysis was performed as previously described (47 (link)). In brief, WBC were isolated from 4T1-tumor bearing mice and incubated in 5 ml glycine quenching buffer for 30 min at RT. The cells were incubated with the LQI tetramer SA-Alexa 568 and CD177-Alexa 647 antibody for 30 min at 4°C. Following washing and fixation (PFA) the cells were stained with YOYO-1. Before imaging, cells were resuspended in STORM imaging buffer (100mM MEA, 10% glucose, Glox (11.2 mg/ml glucose oxidase (Sigma) and 1.8 mg/ml catalase (Sigma)) in dilution buffer (50 mM NaCl, 200 mM Tris in D₂O)). STORM was performed in a Nikon Eclipse Ti-E microscope with a CFI Apo TIRF × 100 DIC N2 oil objective (NA 1.49, WD 0.12 mm) equipped with a Andor iXon-897 EMCCD camera. Super-resolution images were reconstructed from a series of at least 5000 images per channel using the N-STORM analysis module, version 1.1.21 of NIS Elements AR v. 4.40 (Laboratory imaging s.r.o.). Co-localization analysis was performed using the ImageJ plugin ‘Interaction Factor package’ described previously (35 (link)). The calculated ‘Interaction Factor’ scores between 0 and 1, where 0 represents no interaction and 1 represents complete overlap.

HEK-HT stably infected with lentiviruses derived from LentiCRISPR-v2.0-Puro-sgEFR3A-1 and -sgEFR3B were stably infected with lentivirus derived pCDH-GFP-KRAS^G12V-IRES-mCherry-CAAX. 72 h later the cells were plated on 35 mm culture plates. 24 h later cells were placed in a humidifying chamber controlled at 37 °C with 5% CO₂ and live cell images collected using Andor Dragonfly spinning disk confocal microscope using 63 × 1.20 water immersion UPlan S-APO objective using an Andor iXon 897 EM-CCD camera.

PC12 cells transfected with Flag-DOR and either Nb39-mVenus or Venus-miniGsi were plated on poly-D-lysine-coated coverslips and grown at 37°C for 48 hr. To induce intracellular accumulation of newly synthesized DOR, cells were treated with NGF (100 ng/ml) for 1 hr prior to treatment for 15 min with 10 µM β-chlornaltrexamine (CNA; Sigma-Aldrich, #O001) or CNA followed by 10 µM SNC80 (Tocris, #0764) for 5 min. Cells were fixed with 4% paraformaldehyde, pH 7.4, for 20 min at 25°C followed by blocking with phosphate-buffered saline with 5% FBS, 5% glycine, 0.75% Triton-X-100, 1 mM magnesium chloride, and 1 mM calcium chloride. Primary and secondary antibody incubations were performed for 1 hr at 25°C in blocking buffer with anti-Flag-M1 (Sigma-Aldrich, #F3040, 1:1000) conjugated with Alexa-647 (Molecular Probes, #A20186) and anti-TGN-38 rabbit polyclonal antibody (Sigma-Aldrich, #T9826, 1:1000), and goat anti-Rabbit IgG conjugated to Alexa-568 (ThermoFisher, #A-11011, 1:1000), respectively. Cells were washed with blocking buffer without Triton-X-100 after primary and secondary incubations. Coverslips were mounted on glass slides using Prolong Diamond Reagent (Molecular Probes, #P36962). Cells were imaged on a Nikon TiE inverted microscope using a x60/1.49 Apo-TIRF (Nikon Instruments, Melville, NY) objective and iXon +897 EMCCD camera (Andor, Belfast, UK).

Analysis of VE cadherin zone expression was performed on immunofluorescent images we prepared using primary rabbit polyclonal anti-human VE-cadherin antibodies (Cell signaling, Danvers, MA, USA) and secondary goat anti-rabbit Texas Red-conjugated antibody (Sigma-Aldrich, St Louis, MO, USA) in HPAECs, which are better suited for this type of measurement than pulmonary microvascular endothelial cells. Images were acquired using a N-SIM super-resolution system, assembled on a TiE inverted microscope (Nikon, Japan) with a 100× oil immersion lens (NA = 1.49) and an iXon-897 EMCCD-camera (Andor, Ireland, effective pixel size 60 nm). Original images were processed using ImageJ software (Gaussian filtration and background subtraction). Measurements of the width of VE-cadherin expression zone were produced using ImageJ software (function “Measure”). Statistical analysis was performed using Sigma Plot 7.1 (SPSS Science, Point Richmond, CA) and Excel. Sigma Plot 7.1 software was used for graphical data presentation.