Alexa fluor 594 conjugated goat anti mouse immunoglobulin g igg

U251, U251+PTEN or Astrocyte cells were treated with purified 2XMBP-scFv proteins for 24 hours in chamber well slides (Millipore Millicell EZ Slides). Cells were fixed with 1% paraformaldehyde/2% sucrose for 15 minutes at room temperature, followed by 100% methanol for 30 minutes at -20°C, and 50% methanol/50% acetone for 20 minutes at -20°C. Slides were then incubated in permeabilization/blocking solution (10% BGS, 0.5% Triton X-100 in phosphate-buffered saline (PBS)) at room temperature for 1 h. Primary antibody (mouse anti-MBP monoclonal antibody, New England BioLabs, Inc. #E8032S) was diluted 1:500 in permeabilization/blocking solution and used to stain cells at 4°C overnight. The secondary antibody used was Alexa Fluor 594-conjugated goat anti-mouse immunoglobulin G (IgG) (Life Technologies). Three washes with PBS with Triton X-100 and four washes with PBS were each performed after primary incubation and after secondary incubation. Cells were costained with DAPI to visualize the nuclei. Slides were imaged on a Zeiss-CARV II confocal microscope.