Computational protocol: Phosphoinositide 3-kinase signaling pathway mediated by p110α regulates invadopodia formation

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Protocol publication

[…] Cells were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 5 min. To detect the localization of GFP-Akt PH construct and PDK1, cells were fixed and permeabilized in 4% paraformaldehyde, 0.1% glutaraldehyde, and 0.075 mg/ml saponin for 1 h at 37°C. The cells were blocked in 1% BSA and 1% goat serum for 30 min. The cells were incubated with primary antibodies for 1 h and then with fluorophore-conjugated secondary antibodies and phalloidin (Invitrogen) for 30 min. Samples were observed with a confocal microscope (IX81-ZDC-DSU; Olympus) equipped with a cooled charge-coupled device camera (ORCA-ER; Hamamatsu Photonics), and the imaging system was driven by MetaMorph software (Universal Imaging). All images were acquired using 60× (PLAPON60×O; NA 1.42) or 100× (UPLSAPO100×O; NA 1.4) oil objectives. Images were analyzed and processed with various software packages, including MetaMorph, ImageJ (version 1.41o; National Institutes of Health), and Photoshop (CS4; Adobe). […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Breast Neoplasms, Neoplasms