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Immunohistochemistry in human placental tissue--pitfalls of antigen detection
Arnd Honig1, Lorenz Rieger, Michaela Kapp
1Department of Obstetrics and Gynecology, Josef-Schneider-Strasse 4, D-97080 Würzburg, Germany.
Insights
Nonspecific binding of mouse IgG2b antibodies to human placental endothelial cells (EC) can cause misleading research results. Preincubation with human IgG effectively blocks this artifact, ensuring accurate staining for dendritic cells (DC) and other targets.
Area of Science:
- Immunohistochemistry
- Reproductive Biology
- Cell Biology
Background:
- Incongruous staining results are common in placental research, potentially misleading investigators.
- Unexpected CD83 and human leukocyte antigen (HLA)-G antibody staining was observed on placental endothelial cells (EC).
- These antibodies, along with control antibodies, were of the mouse IgG2b isotype.
Purpose of the Study:
- To investigate the cause of nonspecific antibody staining in human placental endothelial cells.
- To develop a method to prevent artifactual staining in placental tissue.
- To ensure accurate detection of dendritic cells (DC) and other targets at the fetomaternal interface.
Main Methods:
- Analysis of human placental endothelial cell (EC) staining with CD83, HLA-G, and IgG2b isotype control antibodies.
- Testing of various blocking techniques for nonspecific antibody binding.
- Introduction and evaluation of a preincubation step with purified human IgG.
Main Results:
- Human placental capillaries showed high-affinity binding for mouse IgG2b antibodies.
- Standard blocking techniques failed to prevent this nonspecific EC staining.
- Preincubation with purified human IgG abolished all nonspecific staining, including for CD83 and HLA-G, without affecting specific signals.
- This blocking is presumed to be via Fc receptors.
Conclusions:
- Mouse IgG2b antibodies bind nonspecifically to vascular EC in the human placenta.
- Blocking with purified human IgG effectively prevents this artifactual staining.
- This blocking method is potentially applicable to other frozen tissues expressing Fc receptors.
Abstract:
Because incongruous controversial staining results are a common phenomenon in the placenta, methodical investigations are important to prevent researchers from obtaining misleading results. While investigating dendritic cells (DC) at the human fetomaternal interface, we observed staining of endothelial cells (EC) in chorionic villi for CD83. Given the high specificity of this antigen for DC, this did not seem credible. Previous studies had revealed the same surprising staining pattern with human leukocyte antigen (HLA)-G antibodies. We therefore analyzed human placental EC staining more closely. Both CD83 and HLA-G antibodies were of the same mouse IgG2b isotype. We also observed EC staining with a panel of control antibodies of the IgG2b isotype. This suggests a high affinity of human placental capillaries for mouse IgG2b. Several commonly used techniques for blocking nonspecific binding of antibodies could not prevent this nonspecific EC staining. A new preincubation step with purified human IgG was introduced. This abolished any placental EC staining with CD83, HLA-G, and IgG2b isotype control antibodies, presumably by blocking Fc receptors, whereas specific staining patterns remained unchanged. Mouse antibody of the IgG2b isotype are bound nonspecifically by vascular endothelial cells in human placenta and this can be overcome by blocking with purified human IgG. This blocking procedure could also be appropriate for frozen tissues other than placenta in which Fc receptors are expressed.
