Alexa fluor 488 conjugated goat anti mouse igg or anti rabbit igg

Cells (1 × 10⁴/well) were seeded on 8-well chamber slides (Lab-Tech) after transfection. The cells were fixed, permeated and blocked. Then, they were incubated with the following antibodies: anti-VIM (1:200, Abcam), anti-ITGAV (1:1000, Cell Signaling Technology), anti-ITGB1 (1:1000, Cell Signaling Technology), anti-ITGB3 (1:500, Cell Signaling Technology) or anti-CD47 (1:200, Abcam). The secondary antibody, Alexa Fluor 488-conjugated goat anti-mouse IgG or anti-rabbit IgG (1:500, Invitrogen), was applied for 1 hour. Slides were mounted with antifading solution containing 4′-6′-diamidino-2-phenyl-indole (Vector Laboratories). Images were taken using a confocal microscope (Zeiss). All experiments were conducted in triplicate.

Antigen retrieval was conducted on the deparaffinized tissue sections with BD Biopharmingen Retreivagen A, according to the manufactures instructions (BD Bioscience, San Jose, CA, USA). The tissue sections were blocked in 4% Marvel dried skimmed milk powder (MPBS) for 1 hr. Then they were incubated with 100 μl of either anti-CD31 mouse monoclonal antibody [P2B1] or rabbit polyclonal von Willebrand Factor (vWF) antibody (Abcam, Cambridge, UK) 1:500 in 2% MPBS under cover glass for 1 hr. The slides were washed two times in PBS and further incubated with 100 μl secondary antibody, Alexa Fluor 488 conjugated Goat Anti-mouse IgG or Anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) 1:40 in 2% MPBS for 1 hr under cover glass. The slides were washed three times in PBS before being mounted with Vectashield inc. DAPI (Vector Laboratories, Burlingame, CA, USA). Visualization was performed with a Leica DMI3000 B Fluorescence microscope (Leica Microsystems, Wetzlar, Germany), and an Olympus DP72 digital camera with Cell^B image acquisition software (Olympus, Tokyo, Japan). A blood vessel harbouring fluorescent positive cells was identified on the tissue, and the location marked underneath the slide with a diamond tip glass cutter pen. This marking allowed for subsequent relocation by brightfield microscopy and proper placement of the shadow stick above target area.