Mouse anti integrin αvβ3 clone lm609

Cells grown on coverslips were fixed with 3% paraformaldehyde and stained with primary and fluorescently labeled secondary antibodies. The following antibodies were used: mouse anti-α-smooth muscle actin (α-SMA) (clone 1A4, Sigma, Darmstadt, Germany), mouse anti-synaptopodin (clone D-9, Santa Cruz, Santa Cruz, CA, USA), mouse anti-cytokeratin 18 (CK18) (clone RGE-53, Merck Millipore, Darmstadt, Germany), rabbit anti-fibronectin (Sigma), mouse anti-integrin α_vβ₃ (clone LM609, Millipore), mouse anti-CD31 (Dako, Glostrup, Denmark), and mouse anti-N protein PUUV (A1C5, Progen, Heidelberg, Germany). Cell nuclei were stained by Hoechst 33342 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Images were taken using an Axiocam 506 mono camera attached to an Axio Observer.D1 inverted microscope (Carl Zeiss, Oberkochen, Germany). For Western blot analysis, the following primary antibodies were used: rabbit anti-PUUV N protein and mouse anti-α-tubulin (Sigma). Loading was verified by the detection of tubulin on the same membrane. Detection was performed by using near infrared fluorescent dye (IRDye)-conjugated secondary antibodies and an Odyssey CLx infrared imaging system (Li-Cor, Lincoln, NE, USA).

For immunofluorescence, cells grown on coverslips were fixed, permeabilized and stained with primary and appropriate fluorescently-labelled secondary antibodies. The following antibodies were used: mouse anti-CD31 (endothelial marker) (Dako), mouse anti-cytokeratin 18 (epithelial marker) (Millipore), mouse anti-PUUV nucleocapsid protein (Progen), and mouse anti-integrin α_Vβ₃, clone LM609 (orthohantaviral receptor) (Millipore). Cell nuclei were stained with Hoechst 33342 (Invitrogen). Images were taken using an Axiocam 506 camera attached to an Axio Observer.D1 microscope (Zeiss). For Western blot analysis, the following primary antibodies were used: rabbit anti-PUUV N protein, rabbit anti-integrin β₃ (orthohantaviral receptor) (Santa Cruz) and mouse anti-α-tubulin (Sigma). Equal loading was verified by the detection of tubulin on the same membrane. Detection was performed by using near infrared fluorescent dye (IRDye)-conjugated secondary antibodies and an Odyssey infrared imaging system (Li-Cor Biosciences).