Microwave stabilization enhances immunocytochemical detection of estrogen receptor in frozen sections of macaque oviduct.
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.
Slayden
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OD
Division of Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton 97006, USA.
Koji
T
T
Brenner
R M
RM
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
D000911:Antibodies, Monoclonal; D004965:Estrogen Antagonists; D004967:Estrogens; D011960:Receptors, Estrogen; D014316:Tritium; D004997:Ethinyl Estradiol; D011374:Progesterone; C011310:moxestrol