Computational protocol: Delta-like 4/Notch signaling promotes ApcMin/+ tumor initiation through angiogenic and non-angiogenic related mechanisms

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Protocol publication

[…] Small and large intestinal tumors were fixed in a 4% (w/v) paraformaldehyde in PBS solution at 4 °C for 1 h, cryoprotected in 15% (w/v) sucrose in PBS solution, embedded in 7.5% (w/v) gelatin in PBS solution, snap frozen in liquid nitrogen and cryosectioned in 10 and 20 μm-thick sections. The following primary antibodies were used: anti-PECAM-1, anti-E-cadherin (BD Biosciences, San Jose, USA), anti-α-SMA, anti-PCNA, anti-Lgr5, anti-HIF1α, anti-Cyclin D1 (Abcam, Cambridge, UK), anti-Dll4 (R&D Systems, Minneapolis, USA), anti-lysozyme (Dako, Glostrup, Denmark), anti-non-phospho (active) β-catenin (Cell Signaling Technology, Danvers, USA). Species-specific secondary antibodies conjugated with Alexa Fluor 488 and 594 (Invitrogen, Carlsbad, CA) were used to reveal primary antibody binding. Tissue sections were incubated with primary antibody overnight at 4 °C and with secondary antibody for 1 h at room temperature. Nuclei were counterstained with 4′, 6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; Molecular Probes, Eugene, OR).Fluorescent immunostained sections were examined under a Leica DMRA2 fluorescence microscope with a Leica HC PL Fluotar 10 and 20×/0.5 NA dry objective, captured using Photometrics CoolSNAP HQ, (Photometrics, Friedland, Denmark), and processed with Metamorph 4.6-5 (Molecular Devices, Sunnyvale, CA, US). Morphometric analyses were performed using the NIH ImageJ 1.37v program. After transforming the RGB images into binary files, the percentage of white pixels per field was defined as a positive signal.Under the effect of 2-2-2 tribromoethanol anaesthesia, biotin-conjugated lectin from Lycopersicon esculentum (100 μg/100 μl of PBS) or 1% Evans’ Blue solution (Sigma, St. Luis, MO, US) were administered in the caudal vein to mark vessel perfusion and extravasation, respectively. Both solutions were allowed to circulate for 5 min before the vasculature was transcardially perfused with 4% (w/v) paraformaldehyde in PBS solution for 3 min. Tumor samples were collected and processed as described above. Tissue sections were stained and tumor perfusion was quantified by determining the percentage of red PECAM-positive structures that were co-localized with Streptavidin-Alexa 488 (Invitrogen, Carlsbad, CA, US) signals. Evans’ Blue is red fluorescent and extravasation was visualized in contrast to green fluorescent vascular structures.Apoptosis was measured using the TUNEL assay (Roche, Mannheim, Germany). […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis
Diseases Adenoma, Neoplasms, Colorectal Neoplasms