Alexa fluor 488 conjugated goat anti rabbit 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 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).

Neonatal rat ventricular myocytes were analysed for cardiac α‐actinin expression by immunofluorescence to assess cardiomyocyte hypertrophy. The cells were washed three times with PBS, fixed with 4% paraformaldehyde, permeabilized in 0.2% Triton™ X‐100 in PBS, and blocked with 8% goat serum for 1 hour at room temperature. Then, the cells were stained with a monoclonal anti‐α‐actinin antibody at a dilution of 1:100 in 1% goat serum overnight. The cells were then incubated with an Alexa Fluor 488‐conjugated goat anti‑rabbit immunoglobulin G (IgG) (Invitrogen, USA) secondary antibody for 1 hour at 37°C. DAPI was used for visualization of nuclei. Images were obtained with a fluorescence microscope (Olympus DX51, Japan). Single myocytes was measured using IPP 6.0. Quantitative data for myocyte size were obtained from >150 randomly selected myocytes in three independent experiments.