Immunofluorescence Analysis of Signaling Pathways in Breast Cancer

BC tumor tissues were collected at the indicated times, snap-frozen in optimal cutting temperature medium (Sakura Finetek), and 8-μm sections were prepared and stained with the following antibodies: rabbit anti pAKT-S473 (1:400), pSMAD3(1:200), pERK1/2 (1:200), AKT (1:400), SMAD3 (1:500), and ERK1/2 (1:400), as described previously^{22 (link),39 (link),42 (link),43 (link)}. Stained sections were analyzed using fluorescence imaging microscopy (Leica) and ImagePro Plus Capture and Analysis software (Media Cybernetics). Positive areas were quantified in 15 independent fields/section using the Image Pro-Plus software^{22 (link),39 (link),42 (link),43 (link)}.

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Wang W., Kansakar U., Markovic V., Wang B, & Sossey-Alaoui K. (2020). WAVE3 phosphorylation regulates the interplay between PI3K, TGF-β, and EGF signaling pathways in breast cancer. Oncogenesis, 9(10), 87.

Publication 2020

optimal cutting temperature medium ImagePro Plus Capture and Analysis software

Antibodies Erk1 Fluorescence microscopy Frozen Microscopy Rabbit Smad3 Tissues Tumor

Corresponding Organization :

Other organizations : University School, Case Western Reserve University

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Variable analysis

independent variables

Time of tumor tissue collection

dependent variables

Phosphorylation levels of AKT (pAKT-S473)
Phosphorylation levels of SMAD3 (pSMAD3)
Phosphorylation levels of ERK1/2 (pERK1/2)
Total protein levels of AKT
Total protein levels of SMAD3
Total protein levels of ERK1/2

control variables

Optimal cutting temperature medium (Sakura Finetek) for snap-freezing tumor tissues
Antibody concentrations: rabbit anti pAKT-S473 (1:400), pSMAD3 (1:200), pERK1/2 (1:200), AKT (1:400), SMAD3 (1:500), and ERK1/2 (1:400)
Fluorescence imaging microscopy (Leica) and ImagePro Plus Capture and Analysis software (Media Cybernetics) for stained section analysis
Quantification of positive areas in 15 independent fields/section using Image Pro-Plus software

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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