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.