Immunofluorescence and immunohistochemistry were performed on formalin fixed, paraffin-embedded tumor sections as described previously (28 (link)). The following antibodies were used for immunofluorescence: HA (1:1000) and STAT3 (1:200) (all Cell signaling, Danvers, USA) and for immunohistochemistry: NFATc2 (1:200, Abcam, Cambridge, UK), p-NFATc2 (S213/217/221) (1:50, Santa Cruz Biotechnology, Santa Cruz, USA), STAT3 (1:100, Cell Signaling), p-STAT3 (Y705) (1:50, Cell signaling), p-GS (S641/645) (1:25, Cell Signaling), Ki67 (1:600, Neomarker, Fremont, USA) and GSK-3β (1:200, Epitomics, Hamburg, Germany), CD45 (BD Pharmingen, 1:100). Immunoblotting was performed using standard methods on uncultured, macrodissected tumor protein lysates or lysates from cultured cell lines. Membranes were probed with NFATc2 (Abcam), p-NFATc2 (S213/217/221) (Abcam), Flag (Sigma-Aldrich), HA, STAT3, GSK-3β, CDK 6, p-STAT3 (Y705), p-GS (S641/645), GS (all Cell Signaling), V5 (Invitrogen) and β-actin (Sigma-Aldrich) antibodies. Densitometric analysis was carried out with ImageQuant 5.1.
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Immunofluorescence and Immunohistochemistry of Tumor Samples
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Actin
Antibodies
Cultured cell lines
Densitometric
Embedded paraffin
Formalin
Immunofluorescence
Immunohistochemistry
Membranes
Stat3
Tumor
Tumor protein
Corresponding Organization :
Other organizations : Philipps University of Marburg, Mayo Clinic, University of Illinois at Chicago, St. Joseph's Hospital and Medical Center
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Variable analysis
independent variables
- Antibodies used for immunofluorescence: HA (1:1000) and STAT3 (1:200)
- Antibodies used for immunohistochemistry: NFATc2 (1:200), p-NFATc2 (S213/217/221) (1:50), STAT3 (1:100), p-STAT3 (Y705) (1:50), p-GS (S641/645) (1:25), Ki67 (1:600), GSK-3β (1:200), CD45 (1:100)
- Immunofluorescence and immunohistochemistry results on formalin fixed, paraffin-embedded tumor sections
- Immunoblotting results on uncultured, macrodissected tumor protein lysates or lysates from cultured cell lines
- Formalin fixed, paraffin-embedded tumor sections
- Uncultured, macrodissected tumor protein lysates or lysates from cultured cell lines
- Not explicitly mentioned
- Not explicitly mentioned
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