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The PCR amplified fragments for each of the strains were then subjected to electrophoretic separation on a 5% acrylamide gel on an ABI Prism automated DNA sequencer and scoring of the fluorescent markers was done using the same DNA analysis workstation (ABI Prism 3100 DNA sequencer).
Detection of phenotypic markers was done by flowcytometry.
Next, immunohistochemical staining for the two markers was done on TMA sections as follows.
Subcloning of the C. albicans auxotrophic markers was done by ligation of AscI-PmeI fragments in AscI-PmeI digested pEB1340.
The LOH analysis with SNP markers was done in 20 grade II and 20 grade IV astrocytic tumors.
Genotyping of all 90 markers was done using the Sequenom MassARRAY iPlex Gold® system (Sequenom, San Diego, CA).
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IF studies for intracellular markers were done by counting an average of about 500 cells/marker.
Detection of polymorphism and genotyping of Type I markers were done on Pop1 by SSCP analysis.
Histopathological grading (Nottingham grading system) and immunohistochemistry evaluation of protein markers were done as part of the routine diagnostic procedure.
The analyses of the inflammatory markers were done in one central laboratory, and blinded duplicate samples were measured for quality assurance.
Detection of polymorphism and genotyping of Type I markers were done only on Pop1 by Single Strand Conformation Polymorphism (SSCP) analysis.
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