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 a s Her-2/neu assessment in mammary carcinoma. Reproducibility of immunostain ing techniques and microscopical evaluation are prerequisites for a standar dized and reliable quantitation of immunostaining intensity. In the present study, different staining and microscopical techniques, including fluoresc ence microscopy, epipolarization microscopy of immunogold-silver, and absor bance microdensitometry were compared concerning suitability for quantitati ve evaluation. We describe a staining procedure using alkaline phosphatase- based immunohistochemistry with the substrate Vector Red acid subsequent mi crodensitometry with a custom-designed absorbance filter. We have character ized linearity of the staining intensity in dependence of development time, antibody concentration, and section thickness by means of artificial stand ards consisting of agarose blocks into which immunogold- or alkaline phosph atase-conjugated antibodies were incorporated. Applicability of the differe nt techniques was tested by anti-CD45 immunostaining of leukocytes within r at lung tissue detected by immunofluorescence, immunogold-silver epipolariz ation microscopy, as well as alkaline phosphatase-based Vector Red absorban ce or fluorescence measurement. Excellent qualities of Vector Red for quant itative microdensitometric evaluation of staining intensity were particular ly obvious for absorbance microscopy. Applicability in paraffin-embedded ti ssue as well as in cryosections, excellent segmentation, linearity over a w ide 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.