Immunofluorescence Analysis of HBMEC and HIBCPP Cells

Immunofluorescence analysis of HBMEC grown in chamber slides and HIBCPP cells cultivated in the inverted cell culture insert model was performed as previously described [29 (link)]. The following antibodies were used: primary antibodies: chicken anti-vimentin (BioLegend, San Diego, CA, USA), goat anti-Met (Abcam, Cambridge, UK), mouse anti-Ecad (BD, Franklin Lakes, NJ, USA), and rabbit anti-ZO-1 (Invitrogen, Carlsbad, CA, USA); secondary antibodies: goat anti-chicken Alexa Fluor^® 594, donkey anti-goat Alexa Fluor^® 594, goat anti-mouse Alexa Fluor^® 594, chicken anti-rabbit Alexa Fluor^® 488, and donkey anti-rabbit Alexa Fluor^® 488 (all Invitrogen, Carlsbad, CA, USA). Nuclei were stained with 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI) (1:50,000 in PBS/1% BSA) (Merck, Darmstadt, Germany). Densitometric analysis of Western blot bands normalized to actin was performed using the ImageJ software 1.53e [48 (link)].