title: Microwave stabilization enhances immunocytochemical detection of estrogen receptor in frozen sections of macaque oviduct.
abstract: We have found that microwave (MW) stabilization greatly improves detection of the estrogen receptor (ER) in frozen sections of rhesus monkey oviduct by immunocytochemistry (ICC). Fresh samples of fimbriae were MW-irradiated, frozen, and then cryosectioned. The frozen sections were also MW-treated and then fixed in a paraformaldehyde-based fixative before ICC processing. A parallel set of samples from each monkey were frozen, sectioned and processed for ICC without any MW treatment. MW stabilization clearly increased immunostaining intensity with either of two ER-specific monoclonal antibodies, namely, H222 and 1D5. The greatest increase was noted in tissues collected from spayed or progesterone-treated animals. An antibody dilution series indicated that MW stabilization increased the sensitivity approximately 20- to 40-fold. In addition, we incubated spayed macaque fimbriae at 4 C in the presence of 10 nM [3H]Moxestrol and then either froze the tissues immediately (non-MW) or treated them with MW. Slide-mounted cryosections of non-MW and MW-treated tissue were then incubated with either a Tris-EDTA buffer (low salt) or the same buffer containing 4 M KCl (high salt). The quantity of [3H]Moxestrol-occupied ER extracted from the frozen sections by each buffer was determined by a sucrose gradient shift assay. The low salt buffer extracted significantly more radiolabeled ER from non-MW sections than from MW-treated sections (P < 0.01), whereas the high salt buffer extracted equal amounts of ER from both the MW-treated and non-MW sections. MW-irradiation enhanced ICC detectability of ER in frozen sections by greatly reducing the amount of ER extracted during the various washes used during normal ICC processing.
authors.affiliation: Division of Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton 97006, USA.
authors.forename: O D
authors.initials: OD
authors.lastname: Slayden
authors.forename: T
authors.initials: T
authors.lastname: Koji
authors.forename: R M
authors.initials: RM
authors.lastname: Brenner
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology; D002923:Cilia / Q000648:ultrastructure; D015925:Cryopreservation; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000648:ultrastructure; D004965:Estrogen Antagonists / Q000494:pharmacology; D004967:Estrogens / Q000097:blood; D004997:Ethinyl Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D005187:Fallopian Tubes / Q000737:chemistry*; D005260:Female; D005629:Frozen Sections; D007150:Immunohistochemistry / Q000379:methods*; D008253:Macaca mulatta; D008858:Microscopy, Phase-Contrast; D008872:Microwaves; D011374:Progesterone / Q000097:blood; D011960:Receptors, Estrogen / Q000032:analysis*; D014316:Tritium
chemical_list: D000911:Antibodies, Monoclonal; D004965:Estrogen Antagonists; D004967:Estrogens; D011960:Receptors, Estrogen; D014316:Tritium; D004997:Ethinyl Estradiol; D011374:Progesterone; C011310:moxestrol