Application of image analysis for quantitative immunoperoxidase detection of IgE containing cells in lymphoid tissues

Methods for the quantitation of antigen or antibody specific cells within lymphoid structures rely on the laborious and often inaccurate counting of stained cells visually under the microscope. To circumvent these problems we used a combination of computer-linked image analysis, with the Quantimet 720 and specific staining for IgE by immunoperoxidase, to produce a relatively easy, accurate method for the quantitation of specifically stained cells within tonsils and lymph nodes. Frozen sections of bovine lymph nodes and tonsils were stained in an indirect immunoperoxidase technique with either rabbit anti-bovine IgE or normal rabbit serum as the primary reagent. Examination of the stained slides on the Quantimet 720 image analyzing computer revealed cells that were sufficiently dark to be considered above the 'threshold level' of background staining. These were counted as positive picture points. A quantitative evaluation of the relative number of IgE containing cells within the particular lymphoid tissue was arrived at by determining the difference between the percent positive picture in the control tissue incubated with normal rabbit serum (background) and in the sample treated with rabbit anti-IgE. Using this method, it was readily possible to quantitate the presence of IgE containing cells in the tonsils of calves; the highest percentage of positive tissue was present in the tonsils of calves exposed to ovalbumin. Image analysis is a useful technique for quantitative evaluation of any immunoperoxidase stained tissues.