Anti cd45 biotin

Sorting of murine pulmonary endothelial cells was as previously described (40 (link)). Briefly, lungs were inflated with digestion buffer (RPMI plus HEPES; elastase [Worthington Biochemical]; dispase; and DNase I [MilliporeSigma]), removed, and incubated in digestion buffer (45 minutes, 37°C). After digestion, the lobes were dissected from the trachea, diced, filtered through a 70 μm strainer, and further digested for 30 minutes in RPMI plus HEPES, liberase (Roche), and DNase I. RBCs were lysed with Türk’s lysing buffer, and digested cells were stained with anti-CD45–PE (clone 30-F11), anti-CD31–eF450 (clone 390), anti-EpCAM–APC (clone G8.8), and 7AAD (MilliporeSigma). Anti-CD45–biotin (BioLegend) and anti-Ter119–biotin (BioLegend) were added to each sample, and EasySep Mouse Streptavidin Rapid Spheres with an EasySep magnet (STEMCELL Technologies) were used for removal of CD45⁺ cells and RBCs prior to sorting on a FACS ARIA-II (BD Biosciences). Transendothelial electrical resistance was measured across confluent human pulmonary artery endothelial monolayers (passage 4–6; Lonza) in medium containing 2% FBS, using an electrical cell-substrate impedance sensing system (Applied Biophysics), as previously described (35 (link)).

5 μm thick OCT microsections from experimental tumors were mounted on glass slides for immunofluorescent labeling. Briefly, after 15’ fixation with 4%PFA samples were washed in PBS-Tween 0.3% and primary antibodies, anti-rabbit eGFP (1:200, Abcam) anti-CD45 Biotin (1:200, Biolegend),anti-F4/80 Alexafluor-647 (1:200, Biolegend), anti-SOX2 (1:200 R&D) and anti-keratin 14 (1:200, Abcam) were supplemented in PBS/BSA 3% (w/v) and incubated O.N. at 4°C. Samples were further washed 3 times in PBS-Tween 0.3% before the addition of secondary antibody. Goat Anti-rabbit AlexaFluor488 1:1000 and Streptavidin AlexaFluor568 1:500 were added and incubated 1h at RT. Slides were then washed and mounted with mounting media ProLong for visualization. Tumors were imaged using a Spinning Disk Confocal microscope (Yokogawa CSU-X1 on Zeiss Axio Observer). The measurement of the distance between eGFP+ cancer cells and F480+ Macrophages was made blindly by 3 independent observers. This distance was quantitatively assessed with ImageJ software in 5 random tumor sections per mice group. Representative images were displayed.