Od. Slayden et al., MICROWAVE STABILIZATION ENHANCES IMMUNOCYTOCHEMICAL DETECTION OF ESTROGEN-RECEPTOR IN FROZEN-SECTIONS OF MACAQUE OVIDUCT, Endocrinology, 136(9), 1995, pp. 4012-4021
We have found that microwave (MW) stabilization greatly improves detec
tion 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 wer
e 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 o
f two ER-specific monoclonal antibodies, namely, H222 and 1D5. The gre
atest increase was noted in tissues collected from spayed or progester
one-treated animals. An antibody dilution series indicated that MW sta
bilization increased the sensitivity approximately 20- to 40-fold. In
addition, we incubated spayed macaque fimbriae at 4 C in the presence
of 10 nM [H-3]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 buf
fer (low salt) or the same buffer containing 4 M KCl (high salt). The
quantity of [H-3]Moxestrol-occupied ER extracted from the frozen secti
ons by each buffer was determined by a sucrose gradient shift assay. T
he low salt buffer extracted significantly more radiolabeled ER from n
on-MW sections than from MW-treated sections (P < 0.01), whereas the h
igh 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 duri
ng the various washes used during normal ICC processing.