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Normal serum was used as controls with each set of stains.
Three slides (six tissue sections) from each tumour were selected for each set of stains such that each slide contained sections a minimum of 300 μm away from the previous slide.
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Next, a second set of staining was performed for PAX2.
For each set of staining experiments, positive and negative controls were used.
The second set of staining was developed by using the ABC kit and 3,3′-diaminobenzidine, followed by dehydration.
For negative controls, the primary antibody was replaced by phosphate-buffered saline in each set of staining.
The vast majority of the cases have a complete set of staining data and clinicopathologic information upon which statistical analysis was performed.
In addition, peroxidase activity derived from the first set of staining was also blocked using alkaline phosphatase/horseradish peroxidase block (SurModics, MN, USA).
Two full sections of the tonsil tissue were used as positive and negative controls for each set of staining, since tonsil had previously shown to have a high CD4 +, CD8+, CD20+ and CD138+ content, by immunohistochemical staining.
Two full sections of tonsil tissue were used as positive and negative controls for each set of staining as tonsil had previously shown to have a high CD68+ content, by IHC.
Because α-SMA and PAX2 antibody were developed in rabbits, an anti-rabbit IgG antibody Fab fragment (Jackson ImmunoResearch, West Grove, PA, USA) was used to saturate all the binding sites created during the first set of staining.
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