Different variations of the
antigen retrieval technique using different retrieval solutions have been evaluated for their effectiveness in restoring the antigenicity of six intranuclear
antigens, each of which is a potentially valuable prognostic
indicator in
formalin-fixed,
paraffin-embedded tissue sections. The results of immunohistochemical staining for
estrogen receptor,
progesterone receptor,
androgen receptor, p53
protein,
proliferating cell nuclear antigen, and
Ki-67 antigen were compared following the different
antigen retrieval approaches. The strongest immunostaining signal with the clearest background was obtained by microwave heating of dewaxed
paraffin sections for 10 minutes in 0.05 mol/L
glycine HCl (pH 3.5) or in
citrate buffer solution (pH 6).
Urea solution, distilled water, and lead
thiocyanate solution yielded improvements with some
antigens, but less consistently and less impressively than
glycine HCl
buffer or
citrate buffer. Following
antigen retrieval nuclear staining was sharply defined and could be achieved consistently in a variety of tissues after
formalin fixation for as long as 7 days. The duration of fixation, however, was an important variable; generally, the longer the fixation time the more vigorous the retrieval procedure required. This study demonstrates the ability to
stain a variety of intranuclear
antigens, which are not readily demonstrable otherwise, in
formalin-
paraffin sections with a high degree of consistency and reproducibility. The availability of methods that are effective in
paraffin sections may facilitate studies of the possible value of these markers as prognostic indicators for predicting the response of major
tumors to different forms of
therapy. This study also provided insight into the basic principles of the
antigen retrieval method, which may be helpful in attempts to develop a more uniformly standardized technique applicable to many different
antigen systems.