Validation of image analysis for enzyme histochemical and immunocytochemical staining

Immunocytochemical and enzyme histochemical analyses of cells and tissues are used to detect changes in the extent of injury and the expression of various molecules. Image analysis quantitation offers an easier, more efficient technique to evaluate these changes. We studied the application of image analysis for evaluating enzyme histochemistry and immunocytochemistry of cells and tissues as a way to assess stroke. Using brain sections, we compared investigator and computer-generated image analysis of 2,3,5-triphenyltetrazolium chloride stained cerebral infarcts in rats subjected to 2 h middle cerebral artery occlusion and 22 h re-perfusion. Both methods documented the infarct volumes with a comparison of means of less then 5%. This suggests no difference between computer- and hand-calculated values. Computer-generated analysis was easier and faster to use. Using endothelial cell monolayers, immunocytochemical staining of a time course of heat shock protein expression was compared to a grading system using fast red chromagen counterstained with hematoxylin. Results demonstrated greater ease and efficiency with computer-generated image analysis compared to other subjective systems of analysis. Image analysis is more useful for detecting small differences in staining, especially when using 3,3-diaminobenzidine as a chromagen. Investigator bias is also reduced using this system. Our comparisons validate the use of this versatile technology to assess more easily both cell and tissues in stroke research.