Automated Quantification of Apoptosis in Tumor Slides

Sections of 4% paraformaldehyde-fixed paraffin-embedded tumor slides were stained with anti-human caspase-3 rabbit polyclonal antibody or rabbit IgG control. An ABC kit (Vector Laboratories, Burlingame, CA) and DAB kit (Vector Laboratories) was used to develop the signals from antibody staining. Slides were counterstained by hematoxylin (Vector Laboratories). Glass slides were scanned with an Aperio ScanScope scanner (Leica Microsystems, Inc., Lincolnshire, IL) and were analyzed with the automated image analysis algorithm Aperio Positive Pixel Count (Leica Microsystems, Inc.) using the default set of parameters. Within each cross-section, tumor cells were distinguished by their higher nucleus to cytoplasm ratio (N/C ratio), which is observed in cancer cells^{13 (link)}. The ratio of strongly positive signals to the total intensity of the detected signals was determined.

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Sharkey C.C., Li J., Roy S., Wu Q, & King M.R. (2016). Two-stage nanoparticle delivery of piperlongumine and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) anti-cancer therapy. Technology, 4(1), 60-69.

Publication 2016

ABC kit DAB kit hematoxylin Aperio ScanScope scanner Aperio Positive Pixel Count

Anti antibody Antibody Cancer Caspase 3 Cells Cytoplasm Embedded paraffin Hematoxylin Human Nucleus Paraformaldehyde Rabbit Tumor Vector

Corresponding Organization :

Other organizations : Cornell University, Massachusetts Institute of Technology

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Variable analysis

independent variables

Anti-human caspase-3 rabbit polyclonal antibody
Rabbit IgG control

dependent variables

Ratio of strongly positive signals to the total intensity of the detected signals

control variables

4% paraformaldehyde-fixed paraffin-embedded tumor slides
ABC kit (Vector Laboratories, Burlingame, CA)
DAB kit (Vector Laboratories)
Hematoxylin counterstain (Vector Laboratories)
Aperio ScanScope scanner (Leica Microsystems, Inc., Lincolnshire, IL)
Aperio Positive Pixel Count algorithm (Leica Microsystems, Inc.)
Tumor cells distinguished by higher nucleus to cytoplasm ratio (N/C ratio)

controls

Positive control: Anti-human caspase-3 rabbit polyclonal antibody
Negative control: Rabbit IgG control

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