Double Immuno-labelling of Proliferating Villous Cytotrophoblasts in Thick Paraffin Sections: Integrating Immuno-histochemistry and Stereology in the Human Placenta

In order to understand the pathological basis of abnormal villous trophoblast development in diseased placentas, the organ must be sampled by non-biased methods and subject to analysis by stereological tools. This approach permits quantification of cytotrophoblast density and syncytiotrophoblast structure including evidence of apoptotic shedding via syncytial knots. The stereological quantification of cells (or their) nuclei requires that each should be unambiguously identified and counted within a defined volume of tissue. A major limitation of such studies at present is the inability to accurately identify and phenotype subsets of villous cytotrophoblasts that either proliferate or are destined to fuse into the overlying syncytiotrophoblast.We describe the development of a novel double immuno-labelling protocol to selectively identify proliferating villous cytotrophoblast cells in human placental villi using thick (25 μm) paraffin sections suitable for stereological quantification. Cytotrophoblast cells were selectively stained using a monoclonal anti-cytokeratin 7 (CK 7) antibody without antigen retrieval, followed by nuclear Ki-67 co-localisation. Both antibodies displayed full depth penetration with sharp, clearly defined staining precipitates and no cross-reactivity. This double immuno-labelling protocol is reproducible, cost effective and time efficient (8 h). Use of a variety of antibodies following antigen retrieval will be a significant advancement in the ability to accurately quantify sub-populations of villous cytotrophoblast in normal and pathological placentas.