This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. PURPOSE: Recently many studies have used immunolabelling and modelbased methods for counting cells in tissue sections. In this study we attempted to validate a model-based counting method with immunolabelling to count cells in mouse glomeruli. We compared the results to a designed-based counting method previously shown to be unbiased. DESIGN METHODS: Seven mouse kidneys were processed for the two different counting methods. Plastic embedded, 1-?m thick sections stained with toluidine blue were used for the design-based, fractionator/disector method. For the model-based, Weibel- Gomez method, we used 3-?m thick paraffin sections labelled with WT1, an antibody to glomerular podocyte nuclei. Podocytes were counted in 10 glomeruli per mouse using each method. RESULTS: The fractionator/disector method resulted in 88?8 (mean?SD) podocytes per glomerulus counted. The immunolabelled, Weibel-Gomez method resulted in 147?27 podocytes counted per glomerulus. A Wilcoxon matched-pair test showed a statistical difference (p=0.02) in the number of podocytes counted using the two methods. CONCLUSION: In this study we showed the model-based Weibel-Gomez method always overestimates the number of cells counted using the unbiased design-based method and thus we were not able to validate this method as an unbiased counting tool. The overestimation of podocyte number may have been caused by: 1) The antibody was not specific enough, 2) Three micrometer sections resulted in over projection error;3) The Weibel-Gomez method is a biased counting method.
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