Quantitative caveats of standard immunohistochemical procedures: implications for optical disector-based designs

Immunohistochemistry is a ubiquitous technique in histology. Often, the goal of such studies is the quantification of some parameter associated with a particular antigen. When used correctly, the optical disector offers a statistically relevant approach to achieve this goal without bias from cell size, shape, or orientation. This three-dimensional counting probe is virtually embedded within the depth of the tissue section, thus avoiding sampling near the cut surfaces of the section, where cells are often lost during the cutting and subsequent processing steps. It follows that the probability that a cell could be immunolabeled should be equal throughout the section depth to correctly employ the optical disector. In this report, we demonstrate that parameters commonly used in immunohistochemistry often leave the middle of the section unlabeled. Furthermore, the degree of incomplete penetration varies among antibodies but can be overcome in some cases by extending the incubation time of the secondary antibody. The detection of this phenomenon in immunofluorescence preparations and the implications of these findings for quantitative stereology using the optical disector are discussed.