Quantitative assessment of relative changes of immunohistochemical staining by light microscopy in specified anatomical regions.

Despite the advent of ever newer microscopic techniques for the study of the distribution of macromolecules in biological tissues, the enzyme-based immunohistochemical (IHC) methods are still used widely and routinely. However, the acquisition of reliable conclusions from the pattern of the reaction products of IHC procedures is hindered by the regular need for subjective judgments, in view of frequent inconsistencies in staining intensity from section to section or in repeated experiments. Consequently, when numerical comparisons are required, light microscopic morphological descriptions are commonly supplemented with analytical data (e.g. from Western blot analyses); however, these cannot be directly associated with accurate structural information and can easily be contaminated with data from outside the region of interest. Alternatively, to eliminate the more or less subjective evaluation of the results of IHC staining, procedures should be developed that correct for the variability of staining through the use of objective criteria. This paper describes a simple procedure, based on digital image analysis methods and the use of an internal reference area on the analyzed sections, that reduces the operator input and hence subjectivity, and makes the relative changes in IHC staining intensity in different experiments comparable. The reference area is situated at a position of the section that is not affected by the experimental treatment, or a disease condition, and that can therefore be used to specify the baseline of the IHC staining. Another source of staining variability is the internal heterogeneity of the object to be characterized, which means that identical fields can never be analyzed. To compensate for this variability, details are given of a systematic random sampling paradigm, which provides simple numerical data describing the extent and strength of IHC staining throughout the entire volume to be characterized. In this integrated approach, the figures are derived by pooling relative IHC staining intensities from all sections of the series from a particular animal. The procedure (1) eliminates the problem arising from the personal assessment of the significance of the IHC staining intensity, (2) does not depend on the precise dissection of the tissue on a gross scale and (3) considerably reduces the consequences of limited, arbitrary sampling of the region of interest for IHC analysis. The quantification procedure is illustrated by data from an experiment in which inflammatory reactions in the murine spinal cord, measured as microglial activation, were followed by IHC after the lesion of the sciatic nerve.