Carv 2 confocal microscope

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.

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 as above and incubated in permeabilization/blocking solution (as above) at room temperature for 1 h. Primary antibodies were diluted 1:500 in permeabilization/blocking solution and used to stain cells at 4°C overnight. The primary antibodies used were rabbit anti-p-Histone H2A.X Serine 139 (Cell Signalling Technologies #9718), rabbit anti-p-53BP1 Serine 1778 (Cell Signalling Technologies #2675), and rabbit anti-cleaved caspase-3 (Cell Signalling Technologies #9661). The secondary antibody used was Alexa Fluor 488-conjugated goat anti-rabbit 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 co-stained with DAPI to visualize the nuclei. Slides were imaged on a Zeiss-CARV II confocal microscope. Images were visualized and quantified using ImageJ. Statistical significance was determined using the unpaired t test (GraphPad Prism).