Abstract
Immunostaining in clinical routine and research highly depends on standardized staining methods and quantitative image analyses. We qualitatively and quantitatively compared antigen retrieval methods (no pretreatment, pretreatment with pepsin, and heat-induced pretreatment with pH 6 or pH 9) for 17 antibodies relevant for placenta and implantation diagnostics and research. Using our newly established, comprehensive automated quantitative image analysis approach, fluorescent signal intensities were evaluated. Automated quantitative image analysis found that 9 out of 17 antibodies needed antigen retrieval to show positive staining. Heat induction proved to be the most efficient form of antigen retrieval. Eight markers stained positive after pepsin digestion, with β-hCG and vWF showing enhanced staining intensities. To avoid the misinterpretation of quantitative image data, the qualitative aspect should always be considered. Results from native placental tissue were compared with sections of a placental invasion model based on thermo-sensitive scaffolds. Immunostaining on placentas in vitro leads to new insights into fetal development and maternal pathophysiological pathways, as pregnant women are justifiably excluded from clinical studies. Thus, there is a clear need for the assessment of reliable immunofluorescent staining and pretreatment methods. Our evaluation offers a powerful tool for antibody and pretreatment selection in placental research providing objective and precise results.
Highlights
Placentation starts with the implantation of the human embryo into the maternal tissue and represents the key for successful pregnancy
Low-Melting Paraffin Embedding of Thermo-Sensitive Electrospun PCL/PLA Scaffolds Thermo-sensitive electrospun PCL/PLA scaffolds were cultured with first trimester placental villi or the human first trimester trophoblast cell line ACH-3P (Hiden et al, 2007)
Antibodies specific for cytokeratin 7 (CK7), E-cadherin, von Willebrand factor (vWF), zonula occludens-1 (ZO-1), CD31, human leukocyte antigen G (HLA-G), Ki-67, leptin, and CD163 needed selected pretreatments to stain positive for their antigen
Summary
Placentation starts with the implantation of the human embryo into the maternal tissue and represents the key for successful pregnancy. Failures in placentation go along with complications in pregnancy such as preeclampsia, intrauterine growth restriction, placenta creta, and gestational trophoblastic disease often resulting in maternal or fetal death (Silva & Serakides, 2016). Research on implantation abnormalities is reduced to investigations of first to third trimester placentas, as pregnant woman are justifiably excluded for ethical reasons. Immunostaining of placenta sections leads to new insights into fetal development and maternal pathophysiological pathways. In this context, immunostaining permits the evaluation of the histological
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