Quantitative Immunohistochemistry for the Investigation of Regulatory Peptides in Health and Disease

Abstract

Immunohistochemical studies often present the problem of quantifying the immunoreactive structures in an objective and reliable way, particularly when examining samples which may present possible changes. The advent of computerized image analysis has made possible the quantitative evaluation of immunoreactive structures identified with chromogenic or immunofluorescent methods. However, there are a number of potential problems which can be encountered during image analysis quantification. Size changes due to fixation shrinkage have been observed during specimen preparation, while tissue cutting can cause compression of the sections. Non-specific tissue autofluorescence can be reduced by the use of counterstaining or filters. Also, during imaging it is important to stabilize the power supply to provide uniform illumination. Video cameras tend to be more light-sensitive at the center of the field than at the edges, resulting in uneven illumination. For analysis, the average image input can be modified by defining a specific area or deleting unwanted signal. Before the image is measured, grey levels need to be defined to discriminate the image intensity, and analysis parameters should be selected. Using quantitative immunohistochemistry, it is possible to provide an accurate assessment of nerve density or of immunostained surface area from images containing a large number of immunoreactive structures. These measurements can be made rapidly.Statistically significant differences may be demonstrated when comparing control, experimental, or pathological tissues.