Anti rabbit igg fab2 alexa fluor 488 antibody

Cultured U87, U87 EGFRvIII, LN229, and U251 cells were fixed with 4% paraformaldehyde in PBS (Santa Cruz Biotechnology, USA) for 20 min at room temperature. Thereafter, the cells were permeabilized with 0.2% Triton X 100 in PBS for 15 min. Non-specific binding was blocked by incubation with 5% goat serum in PBS for 30 min. The following primary antibodies used for immunofluorescence: anti-HOXA13 antibody (Abcam, UK; dilution 1:100), anti-EGFR antibody (Invitrogen, USA; 2 μg/ml), anti-phosphpo-SMAD2 antibody (Invitrogen, USA; 2 μg/ml), anti-SMAD3 antibody (Invitrogen, USA; at concentration of 2 μg/ml), anti-SMAD2 antibody (Invitrogen, USA; 2 μg/ml), and anti-phosphpo-SMAD3 antibody (Abcam, UK; dilution 1:100). The slides were then incubated in the appropriate antibodies in antibody dilution buffer overnight at 4°C, followed by a further incubation at room temperature for 1 h with Alexa Fluor^®488 donkey anti-mouse IgG antibody (Invitrogen, USA; 1:500), Anti-rabbit IgG Fab2 Alexa Fluor^®488 antibody (Cell Signaling Technology, USA; dilution 1:500) or Alexa Fluor^®594 donkey anti-rabbit IgG antibody (Invitrogen, USA; 1:500). The samples were washed and embedded into mounting medium with 4, 6-diamino-2-phenylindole (DAPI; nuclear DNA was labeled in blue; VECTOR, USA). Microscopy analysis was performed using a confocal laser scanner microscope.