Mouse anti cd31

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).

Immunostaining of laminin was performed as described previously [27 (link)]. For immunofluorescent staining of PDCD10, sections were incubated with rabbit anti-PDCD10 (Atlas, 1:65) at 4 °C overnight and then incubated with biotinylated goat anti-rabbit IgG (Dako, 1:400) at 37 °C for 1 h followed by the substrate reaction with FITC-labelled avidin (Dako, 1:400) at room temperature for 1 h. For double-immunofluorecent staining, the following antibody mixtures were applied to the sections: rabbit anti-PDCD10 (Atlas, 1:65) and mouse anti-GFAP (Sigma, 1:200); rabbit anti-PDCD10 and mouse anti-CD31 (Dako, 1:20); rabbit anti-PDCD10 and mouse anti-CD68 (Dako, 1:100); rabbit anti-PDCD10 and mouse anti-PCNA (Dako, 1:200); mouse anti-PDCD10 (Santa Cruze, 1:50) and rabbit anti-caspase 3 (active form) (Cell signaling, 1:400). After incubation overnight, the sections were incubated with the mixture of biotinylated goat anti-rabbit IgG (1:400) and Texas red anti-mouse IgG (H + L) (Vector Laboratories, 1:200) followed by the substrate reaction with FITC-labelled avidin. Counterstaining was performed with Hoechst-33258. The sections were finally analyzed by using a fluorescence microscope with ApoTome System (Zeiss, Axio Imager M2).