Immunohistochemical Analysis of Tumor Biomarkers

H&E staining and immunohistochemistry were performed as described [39 (link)]. The following primary antibodies were used: mouse anti-IK (1:20; Alomone); rabbit anti-Ki67 (1:5000; Abcam); rabbit anti-activated caspase-3 (1:200; R&D Systems); rabbit anti-CD31 (Santa Cruz Biotechnology); mouse anti-HNA (1:100; Millipore). Secondary antibodies were Alexa-488-conjugated goat anti mouse/rabbit (1:500; Invitrogen). Samples were mounted in Prolong Gold with DAPI (Invitrogen). Sections were scanned at 20X using a Zeiss AxioScan.Z1 slide scanner. Images were exported into ImageJ for processing. Brightness/contrast was adjusted using the ImageJ “Auto” function. Density of Ki67⁺ or activated caspase-3⁺ cells, CD31⁺ vessel structures, and metastasis to lungs were measured across scanned images of whole sections, blinded to treatment [39 (link), 40 (link)].

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Thurber A.E., Nelson M., Frost C.L., Levin M., Brackenbury W.J, & Kaplan D.L. (2017). IK channel activation increases tumor growth and induces differential behavioral responses in two breast epithelial cell lines. Oncotarget, 8(26), 42382-42397.

Publication 2017

rabbit anti-Ki67 rabbit anti-activated caspase-3 rabbit anti-CD31 mouse anti-HNA Prolong Gold with DAPI AxioScan.Z1 slide

Antibodies Caspase 3 Cells Dapi Goat Gold Immunohistochemistry Lungs Metastasis Mouse Rabbit Vessel

Corresponding Organization :

Other organizations : Tufts University, University of York

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Variable analysis

independent variables

Treatment(s) applied to the samples

dependent variables

Density of Ki67+ cells
Density of activated caspase-3+ cells
Density of CD31+ vessel structures
Metastasis to lungs

control variables

Brightness/contrast adjustment using ImageJ 'Auto' function
Blinding of treatment groups during measurement

controls

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

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