Chicken anti vimentin

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)].

Rabbit anti-nestin (for immunofluorescence 1:2500, for western blot 1:2000; BioLegend (San Diego, CA, USA, 839801), mouse anti-GFAP (for immunofluorescence 1:100; Merck (Darmstadt, Germany), MAB360; for western blot 1:250; Dako (Glostrup, Denmark), M0761), chicken anti-vimentin (for immunofluorescence 1:1000; used throughout the study; for western blot 1:2000; BioLegend, 919101), rabbit anti-vimentin (1:200; Abcam (Cambridge, UK), ab45939; used for the comparison in Figure 1), rabbit anti-TOMM20 (1:200; Abcam, ab186734), mouse anti-Ki67 (1:50, BD Biosciences (Franklin Lakes, NJ, USA, 550609), goat anti-chicken Alexa Fluor 488 (1:1000; Thermo Fisher Scientific, (Waltham, MA, USA, A11039), donkey anti-mouse Alexa Fluor 555 (1:1000; Thermo Fisher Scientific, A31570), donkey anti-rabbit Alexa Fluor 647 (1:1000; Thermo Fisher Scientific, A31573), donkey anti-rabbit Alexa Fluor 555 (1:1000; Thermo Fisher Scientific, A31572), rabbit anti-GAPDH–HRP conjugate (1:500; Cell Signaling Technology, (Beverly, MA, USA, 3683), goat anti-rabbit-HRP conjugate (1:1000; Cell Signaling Technology, 7074), and horse anti-mouse-HRP conjugate (1:1000; Cell Signaling, 7076) were used. The specificity of the GFAP, vimentin, and nestin antibodies was previously validated, on tissues/cell cultures from mice carrying null mutations in the respective genes serving as negative controls.

To obtain protein extracts for Western blotting, cells were washed with PBS twice and lysed in RIPA buffer after the removal of all excess PBS. Proteins of 10 to 20 µg of protein extract were separated on 4–12% Bis-Tris (Invitrogen, Karlsruhe, Germany) and subsequently transferred onto nitrocellulose membranes via electroblotting. Membranes were incubated for 1 h in blocking solution (5% milk powder solution in dH₂O), and proteins were detected with the following 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). The Immobilon Western Kit (Millipore, Schwalbach, Germany) was used for the visualization of detected proteins.