Computational protocol: Colon Cancer Tumorigenesis Initiated by the H1047R Mutant PI3K

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Protocol publication

[…] At necropsy, the colon was removed, flushed with PBS, opened lengthwise and fixed in 10% buffered formalin for 24 hours. Tissues were then stored in 70% ethanol, processed, embedded in paraffin, and cut into 5 μm sections. Every tenth section was stained with hematoxylin and eosin (H&E) for histological review. Immunohistochemistry was performed by dewaxing and rehydrating paraffin-embedded tissues before antigen unmasking was carried out by boiling the samples for 35 minutes in citrate buffer (pH 6.0). Next a peroxidase quench (Peroxidazed 1, PX9684, BioCare Medical, Concord, CA) and background block (Background Sniper, BS966H, BioCare Medical) were applied before the primary antibody was left on overnight. The secondary antibody (Mach 2 Rabbit HRP-Polymer, RHRP520H, BioCare Medical) was applied for 30 minutes followed by chromogen staining (Betazoid DAB Chromogen Kit, BDB2004H, BioCare Medical). Slides were counterstained with hematoxylin (CAT Hematoxylin, CATHE-H, BioCare Medical) and dehydrated for mounting. The primary antibodies included: anti-pAKT (Ser473) (D9E) (#4060, 1:100, Cell Signaling Technology, Beverly, MA), anti-pRPS6 (Ser 235/236) (#4858, 1:50, Cell Signaling Technology), anti-Ki67 (D3B5) (#12202, 1:400, Cell Signaling Technology) and mouse anti-β-catenin (D10A8) (#8480, 1:200, Cell Signaling Technology). The Ki67 proliferation index was measured as the percent of nuclei staining positive for Ki67 per tumor using ImmunoRatio, an ImageJ plugin (http://jvsmicroscope.uta.fi/sites/default/files/software/immunoratio-plugin/index.html). […]

Pipeline specifications

Software tools ImmunoRatio, ImageJ
Applications Bright-field microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Adenocarcinoma, Adenocarcinoma, Mucinous, Colonic Neoplasms, Neoplasms