Ks 400 image analysis software

For fluorescence microscopy, 293T cells were transfected overnight with GFP, GFP-Casp8p41, GFP-Casp8p41 V150A/L157A, or GFP-Casp8p41 V150E/L157K. Cells were then fixed with 2% paraformaldehyde for 1 h at room temperature and then permeabilized with PBS containing 0.1% Nonidet P-40 and 5% bovine serum albumin. Cells were first stained with Bak (Clone TC-100; EMD Millipore) antibody or mouse IgG control (BD), washed three time with PBS, and stained with a goat anti–mouse PE secondary antibody (BD) before being washed three times with PBS. Slides were prepared with DAPI containing Vectashield (Vector Laboratories). Laser scanning confocal microscopy was performed using a confocal laser scanning microscope (LSM 510; Carl Zeiss). Images were taken at the Mayo Microscopy and Cell Analysis Core facility.
Images were analyzed with KS400 Image Analysis Software (Carl Zeiss). The threshold value was determined and separately set for green and red fluorescence channels. The same threshold values were used for all subsequent images. Areas of green, red, and colocalized pixels were measured and data were expressed as a total percent area of green that colocalized with red.

Density of microvascular profiles was assessed automatically as described previously [31] using an AxioCam MRc connected to an AxioPhot microscope (Zeiss). Images were acquired using a 20x objective (Plan Apochromat, NA = 0.32), resulting in a specimen level pixel size of 0.53 μm. Microvessel density (MVD) was defined as the percentage of microvessel area/total tumor area. All image acquisition and processing was performed using custom written macros in KS400 image analysis software (Zeiss).