Human cytotrophoblast populations studied by monoclonal antibodies using single and double biotin-avidin-peroxidase immunocytochemistry.

Abstract

Single and double biotin-avidin-peroxidase immunocytochemical methods in conjunction with an anti-trophoblast monoclonal antibody 18B/A5 and an anti-HLA-A,B,C monoclonal antibody W6/32 were used to study various human trophoblast populations. Several combinations of peroxidase substrates were tried in the double-labeling procedure. It was concluded that the use of 4-chloro-1 naphthol to develop the primary sequence peroxidase and of 3-amino-9-ethyl carbazole for the second sequence peroxidase was the most suitable. The significant findings were: Monoclonal antibody 18B/A5 proved to be a useful marker for villous as well as nonvillous trophoblast, which facilitated the identification of these cells particularly in the placental bed. The expression of MHC Class I antigens was not confined to extravillous trophoblast but these antigens were also demonstrable on the villous cytotrophoblast proliferating to form new primary villi. Double labeling revealed that many of these cells, particularly those furthest away from the mesenchymal core, expressed both trophoblast and HLA antigens as shown by a mixing of the colors produced by the two reaction products. A large number of these HLA-A,B,C, positive trophoblast cells were found to infiltrate deep into the uterine myometrium. The hypothesis was put forward that these fetal cells could be the ones that are responsible for maternal sensitization.

A CIITA‐independent pathway that promotes expression of endogenous rather than exogenous peptides in immune‐privileged sites

“…Our findings are the first to show that MHC class II molecules can be induced by a CIITA-independent mechanism under pathological conditions in vivo: corneal allograft rejection and cuprizone-induced demyelination of the brain. It has also been shown that MHC class II is expressed on aborted placentas [31,32]. These results indicate that a CIITA-independent pathway to MHC class II expression exists within cells of certain immune-privileged tissues (cornea, brain, placenta) and that expression of MHC class II under these conditions may have immunopathogenic consequences.…”

Glucocorticoid stimulates expression of corticotropin-releasing hormone gene in human placenta.

“…Acetone/methanolfixed cells were allowed to react with anti-cytotrophoblast monoclonal antibody 18B/A5 (kindly provided by Y. W. Loke, Univ. of Cambridge) (7). Avidin-biotin-peroxidase (Vectastain, Burlingame, CA) and 0.05% diaminobenzidine (Sigma) were used to localize bound antibody.…”

Antibody Elution Method for Multiple Immunohistochemistry on Primary Antibodies Raised in the Same Species and of the Same Subtype

“…Other published methods such as microwaving in citrate buffer and enzymatic precipitation of the substrates as a shielding technique (Butterworth et al 1985;Sternberger and Sternberger 1986;Lewis Carl et al 1993;Lan et al 1995;Tornehave et al 2000) were also compared with the present procedure and were found less efficient, mainly because of their increased tendency of damaging the tissue, the appearance of more sensitive detection methods, or too laborious optimizations. In another recently published method, primary antibodies were non-covalently labeled in vitro with monovalent Fab fragments carrying the reporter molecules (Brown et al 2004).…”

“…After elution, the slides were washed abundantly in PBS-Tween and processed for the next immunodetection procedure. In addition, we also chose to compare other methods such as microwaving in citrate buffer to test the efficiency of enzymatic precipitation of the substrates as an antibody shielding procedure (Butterworth et al 1985;Sternberger and Sternberger 1986;Lewis Carl et al 1993;Lan et al 1995;Tornehave et al 2000;Toth and Mezey 2007).…”

“…A heat-mediated antibody stripping procedure such as boiling in citrate buffer has been proposed to remove the antibodies after a first round of IHC (Lan et al 1995;Tornehave et al 2000;Toth and Mezey 2007), but in our experience, it is prone to remove some delicate biopsies from poly-lysine slides, especially if they have already been antigen retrieved by a previous heat-mediated procedure. Precipitation of peroxidase reaction products around tissue-bound antigen-antibody pairs has also been reported to shield them from interaction with subsequent pairs of the same species/IgG isotope primary-secondary antibodies (Butterworth et al 1985;Sternberger and Sternberger 1986). Although this method had not been yet tested together with more sensitive detection procedures to evaluate the degree of actual shielding, it is also limited by the fact that it cannot be applied in fluores-cent labeling IHC.…”