Following transfer, polyvinylidene fluoride membranes were blotted using the following primary antibodies: mouse anti-α-SMA, clone 1A4 (1:1000; Sigma); goat anti-collagen, type I (1 400; Southern Biotechnology, Birmingham, AL); mouse anti-Mmp1 (1 500; Southern Biotechnology); and mouse anti-Gapdh (1 5000; Sigma).
Western Blot analysis was conducted using the following primary antibodies and dilutions: anti-PTEN: 1 1000, ab154812 (Abcam, Cambridge, UK), anti-beta actin 1 2000, A0760 40 (USBiological, Massachusetts, USA) and anti-GAPDH 1 3000, ab9485 (Abcam), anti-CREB 1 250, 48H2 (Cell Signalling, Boston, Massachussets).
Plates were incubated over night at 4 °C using the following primary antibodies: rabbit anti-Nanog (1 500), rabbit anti-Oct4 (1:1000), mouse anti-SSEA4 (1 500), mouse anti TRA-1-60 (1:500) (all from Abcam) and mouse anti-Sox2 (1 500, R&D Systems).
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Immunostaining was performed as previously described using the following primary antibodies and working concentrations.
Immunohistochemistry was performed by the means of the streptavidin-biotin-peroxidase complex method using the following primary mAbs for: CD20, α-SMA and CD31 (Dako, Glostrup, Denmark).
Coverslips were processed for immunofluorescence staining using the following primary antibodies: rabbit anti-nestin (1∶500, Abcys), mouse anti βIII-tubulin (1∶500, Sigma, clone SDL.3D10), rabbit anti-GFAP (1∶500, Dako), rabbit anti-MAP2 (1∶500, Abcam), mouse anti-IKAP/hELP1 (1∶100, BD Biosciences, clone 33).
Paraffin sections of tumors were stained using the following primary antibodies: rat anti-mouse F4/80 (Serotec), monoclonal anti- arginase-1 BD arginase-1 BDLaboraTransduction Laboratories α-smonoclonalle actin (Sigma), monoclonal anti- Ki 67 (Neomarkers), rabbit anti- phosphor-histone H3 (Cell Signaling), rabbit anti-VEGF (Calbiochem) and monoclonal anti- von Willebrandt factor (DACO).
Western blot analyses were carried out on PVDF membranes using the following primary antibodies and dilutions: mouse anti-ILK (1∶200), rabbit anti-Src (1∶200) and rabbit anti-SPARC (1∶500) - all from Santa Cruz Biotechnology, Inc. (Delaware, CA, USA) - and mouse anti-β-actin (1∶1000) from Millipore (Temecula, CA, USA).
Protein measurements and immunoblottings were performed as described [68] using the following primary antibodies: monoclonal mouse anti-syntrophin 1351 [69], anti-γ-tubulin (Sigma) and anti-Cdk5 (Molecular Probes) antibodies; polyclonal goat (gift from David Drechsel, MPI-CBG, Dresden) and rabbit (Molecular Probes) anti-GFP antibodies.
Embryos were fixed and stained according to previously described methods [33], [34], and using the following primary antibodies obtained from University of Iowa Developmental Studies Hybridoma Bank: mAb 22C10 (1∶50), mAb BP102 (1∶50), mAb 2B8 (1∶30), and mAb 1D4 (1D4).
