Comparison of different detection methods in quantitative microdensitometry

Quantitative evaluation of immunohistochemical staining has become a focus of attention in research applications and in pathological diagnosis, such as Her-2/neu assessment in mammary carcinoma. Reproducibility of immunostaining techniques and microscopical evaluation are prerequisites for a standardized and reliable quantitation of immunostaining intensity. In the present study, different staining and microscopical techniques, including fluorescence microscopy, epipolarization microscopy of immunogold-silver, and absorbance microdensitometry were compared concerning suitability for quantitative evaluation. We describe a staining procedure using alkaline phosphatase-based immunohistochemistry with the substrate Vector Red and subsequent microdensitometry with a custom-designed absorbance filter. We have characterized linearity of the staining intensity in dependence of development time, antibody concentration, and section thickness by means of artificial standards consisting of agarose blocks into which immunogold- or alkaline phosphatase-conjugated antibodies were incorporated. Applicability of the different techniques was tested by anti-CD45 immunostaining of leukocytes within rat lung tissue detected by immunofluorescence, immunogold-silver epipolarization microscopy, as well as alkaline phosphatase-based Vector Red absorbance or fluorescence measurement. Excellent qualities of Vector Red for quantitative microdensitometric evaluation of staining intensity were particularly obvious for absorbance microscopy. Applicability in paraffin-embedded tissue as well as in cryosections, excellent segmentation, linearity over a wide range, light stability, and feasibility for permanent mounting and for long-term storage are the outstanding features of this technique for use in routine quantitative evaluation.