Af594 anti rabbit

The cryosections were fixed in in ice-cold acetone for 10 min followed by 3 x 5 min washes in PBS. Then, the tumor sections were blocked with 10% goat serum (in PBS) for 1 h and incubated for 30 min with Crystal A MausBlock reagent (Innovative Diagnostik-Systeme, Hamburg, Germany) at room temperature, washed 3 x 2 min with PBS. Following, the sections were incubated with Crystal B MausBlock reagent (Innovative Diagnostik-Systeme, Hamburg, Germany) for 5 min at room temperature and washed again 3 x 2 min in PBS. Afterwards, the slides were incubated overnight at 4 °C with mouse MAB1 (1:50, Hydroxyprobe Inc., MA, USA), rat CD31 (1:20, Dianova Research, Hamburg, Germany) and rabbit Carbonic Anhydrase 9 (1:100, Abcam, UK) antibodies diluted in PBS containing 0.1% BSA plus 1% goat at serum plus 0.1% Tween-20. Subsequently, the slides were washed 3 x 5 min with PBS and then were incubated with AF488 anti-mouse, AF594 anti-rabbit and AF680 anti-rat antibodies (1:500, ThermoFisher Scientific, MA, US) diluted in 1% goat serum for 1 h, at room temperature. Then the slides were washed 3 x 5 min in PBS and mounted with coverslips using Prolong Diamond mounting agent (ThermoFisher Scientific, MA, US).

After 3-h adherence on RGD-membrane, cells were treated firstly with 4% paraformaldehyde and then 0.1% Triton X-100 treatment for 20 min each at 37 °C. 5% BSA with 10% normal donkey serum (blocking buffer) was used for overnight blocking at 4 °C. Samples were then incubated with the primary antibody in blocking buffer at 4 °C for 48 h. Primary antibody was then washed with PBS, followed by secondary antibody incubation for 2 h in room temperature. Primary antibodies included activated human integrin-β3 antibody (1:25, anti-LIBS2 epitope clone ab62, Millipore MABT27) and pFAK-Y397 (1:50, Thermo Fisher Scientific 44-624G). Secondary antibodies included AF594-anti-mouse (1:1000, Thermo Fisher Scientific A-21203) and AF594-anti-rabbit (1:1000, Thermo Fisher Scientific A-21207).