0022-1554/89/$3.

30
The Journal of Histochemistry and Cytochemistry Vol. 37, No. 8, pp. 1207-1213, 1989
Copyright © 1989 by The Histochemical Society, Inc. Printed in U.S.A.

Original Article

New Freeze-Dry and Vapor Fixation Method for

Immunohistochemistry of Soluble Proteins: Subcellular
Location of the Progesterone Receptor

A. PEKKI and P. TUOHIMAA’

Department ofBiomedical Sciences, University of Tampere, 33101 Tampere, Finland

Received for publication October 14, 1988 and in revised form February 22, 1989; accepted March 1. 1989 (8Al50l).

We describe a new application of freeze-drying and vapor the present method, both unoccupied and occupied PR were
fixation for immunohistochemical location of soluble pro- located in the nudei, a similar finding as with the earlier
teins. The method avoids the liquid phase, which eliminates liquid fixation method. The results further support the con-
the possible diffusion of soluble proteins. Two vapor fixa- cept that PR is an intranuclear protein independent of its
tives, paraformaldehyde and p-benzoquinone, were tested ligand occupation. PR was detected in a few cells inside the
and p-benzoquinone was found to preserve antigenicity of follicles of the bursa of Fabricius and in the smooth muscle
progesterone receptor (PR) and ovalbumin better than para- cell nudei of the small intestine, observations not previously
formaldehyde. The method proved to be highly sensitive, made owing to the insensitivity of the earlier methods. (J
since higher concentrations of antigen were found in some Histochem Cytochem 37:1207-1213, 1989)
tissues and some tissues found to be antigen negative by ear- KEY WORDS: Immunohistochemistry; Progesterone receptor; Freeze-
tier liquid fixation methods proved to contain antigen. The dry; Vapor fixation.
location ofPR as a highly soluble protein was studied. With

Introduction tors have been shown to be located unoccupied in the cytosol and
occupied in the nucleus (Gorski et at. , 1968; Jensen et at. , 1968;
Histochemical procedures such as fixation may involve severe in-
Noteboom and Gorski, 1965). From these studies it was concluded
terference by artifacts in the immunohistochemical location of solu-
that steroid receptors transtocate from cytoplasm to nucleus by ligand
ble proteins. Cytoplasmic or secretory proteins can be transferred
occupation. However, enucleation studies indicated that receptors
into the nuclei by certain fixatives. Aldehyde fixatives cause an
might subsist in a concentration equilibrium between cytoplasm
artificial nuclear location of cationic lactoferrin (Konttinen and
and nucleus (Welshons et al., 1985; Sheridan et at., 1981). The latest
Reitamo, 1979). Similarly, other cationic proteins, such as avidin
immunohistochemical studies appear to indicate that both unoc-
and steroid receptors, have a high affinity for nucleic acids owing
cupied and occupied receptors are intranuctear except for their syn-
to their basic charge (Grody et at., 1982; Bayer and Wilchek, 1980).
thesis, which is perinuclear (Isota et at., 1986; Gasc et at., 1984;
On the other hand, nuclear proteins may transtocate into the cyto-
Greene et at. , 1984). There are, however, also immunohistochemi-
plasm, especially if membranes are damaged by treatments such
cat studies in agreement with the classical two-step receptor model
as the Triton X-100 commonly used to improve the penetration of
(Agnati et al., 1985; Raam et al., 1982). Part of the differences in
antibodies (Isola et at., 1987). Another problem with liquid fixa-
the results might be explained on the basis ofmethodological varia-
tion procedures is the relatively poor penetration of fixatives into
tions. All the methods used can be criticized in that they use aque-
tissue pieces. Therefore, soluble proteins can artificially attach to
ous fixation methods, which could cause artificial diffusion of steroid
any charged domain in a damaged tissue piece after dissection and
receptors from cytoplasm to nucleus or vice versa. In this study we
before proper fixation. Because mobility of soluble proteins can
used chicken progesterone receptors as a model to test the method.
occur in the soluble phase before fixation, we sought to avoid the
aqueous phase by a rapid freezing in liquid nitrogen.
The subcellular location ofsteroid receptors has been a subject
Materials and Methods
of debate during recent years. On the basis of biochemical studies
Animals and Treatments. Immature White Leghorn chicks (age 2-6
using tissue homogenization and ultracentrifugation, steroid recep-
weeks) were used. The chicks were treated with 173-estradiol 3-benzoate
(Sigma Chemicals; St. Louis, MO) dissolved in sesame oil (1 mg/animal/day)
1 Correspondence to: P. Tuohimaa, Dept. of Biomedical Sciences, Uni- or used untreated. First they received seven daily injections. Thereafter, chicks
versity of Tampere, Box 607, 33101 Tampere, Finland. were without treatment for 1-2 weeks (an estrogen withdrawal period), af-

1207
1208 PEKKI, TUOHIMAA

tel which part of them received a single injection of progesterone (Merck; and 10 p1 biotinylated peroxidase in 1.6 ml PBS, incubated for 30
Darmstadt, FRG)(20 mg/kg body weight). The chicks were killed 1 or 2 hr mm).
later. Pieces ofoviduct, bursa ofFabricius, and intestine were dissected out 9. Wash twice in PBS for 10 mm each.
as quickly as possible and immediately frozen in liquid nitrogen. The dis- 10. Incubate with 0.025%, 3,3’-diaminobenzidine-HCI (Sigma) and
sected pieces were from 2 mm to 5 mm in diameter. After excision the 0.03% hydrogen peroxide in 0.5 M Tris-saline for 6-10 mm.
specimens were placed on rigid pieces of paper with a label. 11. Termmnate the reaction in 0.5 M Tris buffer, pH 7.6.
12. Dehydrate sections and mount in Entellan (Merck).
Freeze-Drying. The methodology for the conventional Falck-Hillarp
freeze-dry technique has been described earlier (Er#{228}nk#{246}.
1967; Fatck and Where necessary, endogenous peroxidase was blocked with an incubation
Owman, 1965). A “cold finger” type of freeze-drier with a two-stage me- in 0.5% hydrogen peroxide in absolute methanol for 10 mm after step 2.
chanical pump in combination with an oil-diffusion pump (Edwards high Hematoxylin-eosin staining was performed for morphological analysis of
vacuum; BOC Ltd. Crawley, UK) provides a vacuum of about 10 mbar. the tissue components.
The desiccant baskets containing di-phosphorous pentoxide (Merck) are
placed over the tissues. At this point the temperature should be -40’C. PR Antibodies. The potyclonal antibody IgG RB-S was raised against
and the tissues will rapidly equilibrate to this temperature. For very small the affinity-purified chick oviduct PR component. The specificity of IgG
pieces of tissue, 4-5 days in the freeze-drier is sufficient to ensure good
RB-S was tested by immunoprecipitation, immunoblotting. density gra-
dient ultracentrifugation, and protein A-Sepharose assay methods (Tuo-
drying. Larger pieces may require as long as 10-14 days.
Before removal of the tissues from the freeze-drying apparatus, the tern- himaa et at., 1984). The antibody was highly specific for progesterone binding

perature of the pieces must be raised well above the ambient. The tissue components ofchicken PR. Negligible crossreactivity was observed with other
steroid receptors or with PR ofother animal species studied. The specifici.
holder is transfrrred to warm water without breaking the vacuum and warmed
to room temperature. The vacuum is released immediately before fixation. ties of the mouse monoclonal antibodies PR6, PR13, and PR22 have been
tested by immunoprecipitation, immunoblotting, glycerol gradient cen-
Vapor Fixation. The second step in the Falck-Hillarp method was va- trifugation, photoa.ffinity labeling, and protein A-Sepharose assay (Schneider
por fixation of the dry specimens. In this study we used two fixatives, gas- et at., 1988; Sullivan et at., 1986).
eous paraformaldehyde (Merck)(Er#{228}nk#{246},
1967)orp-benzoquinone (Pearse
and Polak, 1974). The tissue holder was transferred to a desiccator with Controls. Specificity checks were as follows: (a) primary antibody was
substituted with equal concentrations of irrelevant antibody (monoclonal
paraformaldehyde powder in the glass container. A relative humidity of
anti-hLH; Mcdix Biochemica, Finland) or phosphate buffer; (b) the pri-
50-80% is recommended, but suitable humidity must be determined em-
mary antibody was pre-saturated with purified component B ofPR. A five-
pirically for each tissue. For paraformaldehyde the temperature we used
fold excess of PR-B was mncubated with the antibody at 37’C for 4 hr.
was 60 or 80’C and the fixation time was usually 1 or 2 hr. The ratio be-
tween water content of paraformaldehyde and temperature has been de-
scribed by Er#{228}nk#{246}
(1967). When the temperature is lowered, the water con- Results
tentof the paraformaldehyde powder must be increased.
The other fixative wasp-benzoquinone (1-3%) dissolved in toluene at
Freeze-drying of larger pieces was not always satisfactory. Ice crys-
60C for 3 hr in a desiccator (Pearse and Polak, 1974). tits forming during freezing ofthe tissues apparently create empty
spaces in the specimen. Since the size of the crystals increases as
Immersion Fixation. Baker’s fluid fixation was used to compare vapor
the cooling rate decreases, the maximum cooling rate is desirable.
fixation and liquid (immersion, aqueous) fixation. Small pieces of oviduct
However, ice crystal artifacts are often manifest at higher magnifi-
were fixed in Baker’s fixative (10% paraformaldehyde, 1% CaCl2, pH 6.7)
cation in frozen specimens more than 3 mm thick. The use of small
for 2 hr at 0C. Tissues were processed further as described previously (Isola
et at., 1986). pieces of tissues (less than 2 x 2 x 2 mm) has the additional ad-
vantage ofshortening the drying time. Temperatures for vapor fix-
Embedding. The specimens were embedded in dc-gassed paraffin as
ation were 80’C or 60’C. The lower temperature gave the more
soon as possible after vapor treatment, thus avoiding condensation of at-
favorable result, antigenic determinants being easily destroyed at
mospheric water vapor on them.
the higher temperature.
Immunohistochemistry. Sections were cut (2-4 pm thick) and processed The preservation of antigens was better with p-benzoquinone
according to the immunoperoxidase technique, using a biotinylated sec- vapor fixation than with paraformaldehyde vapor, which is demon-
ondary antibody and the avidin-biotin-peroxidase complex (Vectastain re-
strated for ovalbumin in Figure 3. The cell morphology with vapor
agents; Vector Laboratories, Burlingame, CA) at room temperature as follows:
fixation was not as well preserved as with aqueous fixatives, when
1. Deparaffinize in xylene (three times) and rehydrate in graded at- the fixations were made from the same sample (Figures ic and 2a),
cohols. but the nuclear location ofPR was always manifest. A similar result
2. Wash twice in PBS, pH 7.0, for 5 mm each. was obtained with all four antibodies, PR6, PR13, PR22, and IgG
3. Incubate in 3-10% normal rabbit or horse serum according to 5cc-
RB-S. In the chick oviduct the progesterone receptor was found
ondary antibody or in 1% milk diluted in PBS for 15-30 mm.
in epithelial, glandular, smooth muscle, mesothelial (peritoneal),
4. After removal of excess serum, add the primary antibody for 30
and mesenchymal cells (Figures la-id and 2d). The PR was located
mm to 2 hr at room temperature or overnight at 4’C. Final concen-
in the nucleus of the target cells independent of occupation. In
trations of PR6 and PR13 were 0.02-2 pg/mI in 1% normal serum
or milk. the progesterone-treated oviduct (Figure ib), a clearly weaker im-
5. Wash twice in PBS. munostaining was found than in those without progesterone treat-
6. Incubate with the secondary biotinylated antibody at a dilution ment (Figure la). Weak cytoplasmic staining was seen in a few cells,
of 1:100 in 1% normal serum or milk for 30 mm. possible due to a new synthesis of PR in the endoplasmic reticu-
7. Wash twice in PBS. turn caused by estrogen treatment (Isola et al. , 1986). There was
8. Add prepared avidin-biotin-peroxidase complex (10 p1 avidin DH considerably less cytoplasmic staining after progesterone adminis-
 #{149}
..
. ..

.
, ‘ ) I..
r
.
,
/.
\

-
‘

. .- --I.’. ‘
:
.
‘ ‘:

. #{149}‘
#{149}#{149}‘.‘.‘:

#{149}
.
.
-j

. ..
‘J

.,-
. -:.

4
. . J- .
‘ ‘ ‘)..\J
. I ‘ _ \.t . . .,g ‘b., . f : .. -.‘ _ : ‘ .
. . _.:; .‘:- ::d.;;: .‘w: #{149}. . ...

I t\
)
,1.:
,.
:
-)
1
, ‘I I
i;i
Lk_

...... ,
- ‘.‘, .. ;e
:\
. .. . .- ‘ #{149}‘#{149} .

I , /‘,, I

, =‘l_ ‘ A *.

‘:\ : I

,
-I 7/ : .
.. - .i._ ,. : * .

7 ‘ \

_1e -. ‘ ‘I,-. I __ if :‘=‘ ‘: g

Figure 1. Location of PR in the chick oviduct. PR is in the nuclei of epithelial, glandular, and stromal cells. All the samples are freeze-dried and vapor-fixed in
p-benzoquinone. Oviducts treated with estrogen (a) and estrogen and progesterone (b) are stained with monoclonal antibody PR6. Oviducts treated with estrogen
(C) and estrogen and progesterone (d) are stained with monoclonal antibody PR13. (e) No staining is found in control sections when the primary anlibody PR6
is substituted with phosphate buffer. (1) When the primary antibody PR13 is pre-saturated with purified component B of PR there is no staining. e, epithelium;
g, gland; m, smooth muscle cell; s, stromal cell. Bars = 20 pm.
1209
 . . *

, .
. .

.. :
.; .
.it,.,

..3
a
A :
. : #{149}
. #{149}li
a ‘%
, . . - ‘.. ‘ ,.
I
. V .
a
‘S I. .

. .

.‘lI.lu,
#{149}

.
A #{149}#{149} #{149}
0

a
.. ..
.1

*_ .- 4

2b - - I
#{149}
t 1r:v \
. , . .. :_T_
V ‘ ‘#{149}

s;

. ..“ %. ‘- ., \p :; 4 ‘ \‘ - ‘ .i,. . . : “b

: .

#{149}d4P.k!.1: :r’#:
‘t,\
-.. . *w- .: -.

: , ‘ . ‘ . .‘ ‘-‘‘: b.A1 . .-
.- :- .
#{149}
. ..

, ,... . - ,I=’

k\:’ m- L .1*4umt% : :-..4:#

a .. . ..

. ‘‘\#{149} - . ft - . - . . ,.P 4

. ‘‘ . .. . -

,S ‘ \\“#{149}h
1#{188}’h%:, : . - ;. . . .

.‘

*_ . .
,
. . . . . ,- -:
I ..
-

. _s ‘4

‘-.

..

- .*Ot
.,
\: ., 2d1.. ..

-
.

-“
;_

--- --

Figure 2. (a) Location of PR in lhe same estrogen-treated chick oviduct as in Figure ic, using Baker’s fluid fixation and monoclonal antibody PR13. (b) In the
bursa of Fabricius PR is located in the nuclei of interfollicular cells and a few follicular cells. The section is fixed with p-benzoquinone vapor and stained with
monoclonal antibody PR6. The nuclei of smooth muscle cells of the oviduct (C) and small intestine (d) stained with monoclonal antibody PR6, when they are
fixed with p-benzoquinone vapor. i, interfollicular cell; f, follicular cell. Bars = 20 pm.

1210
 -

- . .- - fl:-- - : -
-

e .. : .

.. I
e . .

.
. ,
. *
i-i,
. .. .
‘.. -- % #{149},: _‘ .
. . ,.
,-.. . . ‘- ....-“

-
. - . .1-
. s’-.:#{149}, _ .;.,q
‘ . I #{149}-,. #{149} #{149}
.

. I f .,__

iIT. ‘-‘a..,’
1 : .

: ‘ ‘ #{149}
‘. .

., . ‘I
‘ . . .
=-a,1 ;, ;:t I.

f
4t;: =

. C a

‘‘:. ‘ . :..
. . . -: .. =..-

a
-4 -- , .
,-- - . a’.-..
I -.
_.
. a

-3d a .
.1’ ,
,., .- S

Figure 3. Location of ovalbumin in freeze-dried and vapor-fixed chick oviduct. In oviducts fixed with p-benzoquinone (a,b,d) ovalbumin is preserved better than
those fixed with paraformaldehyde (C). Ovalbumin is mainly located in the cytoplasm of the glandular cells (a,b,c) and a few epithelial cells (a,b). There is no
staining in the control section when the ovalbumin is substituted with PBS (d). Bars = 20 pm.

1211
1212 PEKKI, TUOFHMAA

tration (Figure ib). PR antigen is not usually well preserved in glan- chamber at 6080*C, appreciable shrinkage ofthe tissue piece oc-
dular cells after liquid fixation techniques, especially in the deeper curred.
parts of the tissue, whereas with freeze-dry vapor fixation techniques
a more even distribution of PR can be found throughout the tis-
sue. This might be owing to poor fixation by liquid fixatives of Fixation Mechanisms
glandular cells containing high concentrations ofsecretory proteins. In this study two different types ofvapor fixatives were successfully
In the bursa ofFabricius, PR was found in most ofthe interfol- used. Paraformaldehyde vapor, even at the lower temperature, al-
licutar cells and in a few cells in the medulla and cortex of the folli- most completely destroyed the antigenic determinants of PR and
des (Figure 2b). The type or origin of the follicular receptor- ovalbumin. so it was not a satisfactory fixative for these antigens.
containing cells is not known. Epithelial cells of the bursa did not However, other antigens, such as 90 KD heat shock protein, lac-
contain the antigen. The response of PR to progesterone in the bursa toferrin, and inhibin, do not lose their antigenicity in formalde-
was similar to that in the oviduct. hyde vapor (manuscripts in preparation).
Smooth muscle cells of the intestine contained PR in both Paraformaldehyde is depolymerized during heating to form
progesterone-untreated and -treated chicks (Figures 2c and 2d). formaldehyde gas, which undergoes specific condensation reaction
The PR concentration varied significantly from one muscle cell to with primary and secondary amines. The water content of the va-
another. The sections stained with anti-LH antibodies showed no por is essential for proper fixation, which can be adjusted with sul-
staining, nor did pre-saturation with purified PR of the specific furic acid. However, if the water content is too high a diffusion
antibodies used produce any staining. of antigens may occur (Hamberger et al., 1965). It is possible to
Ovalbumin was found in the cytoplasm of the glandular cells use lower temperatures with paraformaldehyde fixation when the
in the chick oviduct (Figures 3a-3c). Also, some cells in the epithe- water content is increased, and the result is often better because
hum were stained (Figures 3a and 3b). In the control section there some proteins are easily destroyed at high temperatures. Cross-
was no staining (Figure 3d). linking of proteins is apparently the main mechanism of parafor-
maldehyde vapor fixation.
It has been shown thatp-benzoquinone improves the preserva-
Discussion tion of peptide antigens (Pearse and Polak, 1974). Benzoquinone
Although the freeze-drying technique (Falck et at., 1962) and va- and other quinones are believed to react with protein amino groups
por fixation (Pearse and Polak, 1974; Er#{228}nkO,
1964) were developed to form di-substituted quinones, the pH optimum ofthe reaction
years ago the methodology has not been widely applied in immuno- being 8.0 (Means and Feeney, 1971). According to Morrison et al.
histochemistry. In the present study this approach was adopted for (1969), these can further react with either free amino groups or
the location ofa highly soluble protein, progesterone receptor, in sutfydryt groups, but only if the latter are in close proximity. The
various tissues. It was shown that the method ensured good preser- authors found that little intermolecular cross-linking took place
vation of antigenicity and apparently less diffusion of the protein in their in vitro experiments with proteins. It appears that p-ben-
than the earlier liquid-phase methods. Furthermore, the freeze- zoquinone blocks the proteotytic activity of enzymes reacting at
drying and vapor fixation method provides satisfactory preserva- Arg-Lys or Lys-Lys bonds. It is possible, therefore, that one reason
tion of tissue structure at the tight microscopic level. The method for the improved preservation of peptide antigens by bifunctional
can be applied for routine clinical purposes, although it is some- reagents demonstrated by Pearse and Potak (1974) is that they are
what more laborious and more time consuming than routine im- thereby rendered impervious to proteolytic digestion. The good
mersion fixation. It appears that the method may be an alternative results with this vapor fixation may derive from a low degree of
in immunohistochemistry. For electron microscopy the method may cross-linking of proteins, leaving antigenic determinants available
not be optimal for the ultrastructure. However, Pearse and Polak for antibodies. Ovatbumin is an abundant protein and therefore
(1974) have found with p-benzoquinone a good preservation of it is difficult to fix. However, satisfactory fixation was obtained with
electron microscopic structure, although antigenicity survived only p-benzoquinone. It appears that p-benzoquinone is a valid vapor
partially. fixative for several antigens (manuscripts in preparation).

Freeze-Drying Location ofDzffusible Antigens

In this study we used a “cold-finger” type offreeze-drier, on whose The present method avoids the liquid phase, which eliminates the
surface part of the vaporized water is condensed while part is ab- possible diffusion ofsolubte proteins. The ordinary modes of fixa-
sorbed in phosphorous pentoxide. A vacuum, provided by a dual tion have some major disadvantages. Soluble substances are lost,
pump system (a rotation pump and a diffusion pump developing e.g. , lipids in fat-solvent fixatives, proteins, mucoproteins, poly-
i#{248}-
mbar pressure), is maintained around the solid tissue pieces saccharides, and inorganic materials in aqueous fixatives. The cell
to allow vaporization of water. constituents may be displaced by diffusion. To this category be-
Ifthe temperature ofthe pieces is not raised above the ambient tong the so-called streaming artifact and other less obvious types
after freeze-drying, atmospheric water vapor will condense on the of fixation artifact, including aggregation of small particles into
specimen and ruin it (Er#{228}nk#{246},
1967). Short exposure to moist at- granules and clumping of nucleic acids in the nuclei. Denatura-
mospheric air did not appear to have any harmful effect, but if tion of proteins causes alterations in their physical properties and
the cold tissue was transferred (at 40*C) directly into the vapor in their antigenicity (Pearse, 1960). It is therefore possible that solu-
IMMUNOHISTOCHEMICAL METHOD FOR SOLUBLE PROTEINS 1213

ble proteins can show artificial subcellutar location when a liquid tibodies to distinct receptor components. J Cell Biol 99:1193
phase is used in tissue processing (Isola et al. , 1986; Konttinen and Gorski), Toft D, Shyatama D, Smith D, Notides A (1968): Hormone recep-
Reitamo, 1979). However, we recommend use ofthe present method tors: studies on the interaction ofestrogen with the uterus. Rec Prog Horm
whenever the cellular and intracellular location of a new antigen Res 24:45

is analyzed for the first time. Greene G, Sobel N, King W,Jensen E (1984): Immunohistochemical studies
of estrogen receptors. ) Steroid Biochem 20:5 1
Grody W, Schrader W, O’Malley B (1982): Activation, transformation and
Subcel/ular Localization of PR subunit structure of steroid hormone receptors. Endocrine Rev 3:141

Steroid receptors have been variably found in the target cell nuclei Hamberger B, Malmfors T, Sachs C (1965): Standardization of paraformalde-
and/or cytoplasm or in the nuclei only by immunohistochemical hyde and of certain procedures for the histochemical demonstration of
catecholamines. J Hmstochem Cytochem 13:147
methods (Isola et al., 1986; Agnati et at., 1985; Greene et al., 1984;
Morel et al., 1984; Raam et al., 1982). Alt previous approaches in lsolaJ, Pelto-Huikko M, Ylikomi T,.Tuohimaa P (1987): Immunoelectron
microscopic localization of progesterone receptor in the chick oviduct.)
the immunohistochemicat location of steroid receptors have uti-
Steroid Biochem 26:19
lized liquid fixation methods, which can be criticized in the above-
IsolaJ, Ylikomi T, Tuohimaa P (1986): Nuclear origin ofprogesterone receptor
mentioned respects. The present method is basically different and
ofthe chick oviduct cytosol: an immunoelectron microscopic study. 1-listo-
is not vulnerable to the same kinds ofdiffusion artifacts. However, chemistry 86:53
findings similar to those in the present study have also been ob-
Jensen E, Suzukm T, Kawashima T, Stumpf W, Jungblut P, DeSombre E
tamed with liquid fixation methods when the conditions have been (1968): A two-step mechanism for the interaction ofestradiol with rat uterus.
carefully controlled (Isola et at., 1986). This suggests that PR is an Proc Natl Acad Sci USA 59:632
entirely nuclear protein independent of occupation. This concept Konttinen Y, Reitamo S (1979): Effect of fixation on the antigenicity of
is further supported by the finding that PR antigen and ligand human lactoferrin in paraffin-embedded tissues and cytocentrifuged cell
binding show similar nuclear location in freeze-dried unembed- smears. Hmstochemmstry 62:55
ded tissue sections of a target organ (Ylikomi et at. , 1987). The Means G, Feeney R (1971): Chemical modification of proteins. San Fran-
present data thus call into question the hypothesis of the cytoplas- cisco, Holden-Day, 129
mic location of PR and the nuclear translocation of steroid recep- Morel G, Dubois P. GustafssonJ, Radojcic M, Radanyi C, Renoir M, Baulieu
tors by ligand occupation. E (1984): Ultrastructural evidence of progesterone receptor by immuno-
histochemistry. Exp Cell Res 155:283

Acknowledgments Morrison M, Steele W, Danner D (1969): The reaction of benzoquinone

with amines and proteins. Arch Biochem Biophys 134:515
We are very grateful to David Toft, University ofRochester, for providing
us with the PR monoclonalantibodies usedin this study, andto DrMarkku Noteboom W, GorskiJ (1965): Stereospecific binding ofestrogens in the
Pelto-Huikko for stimulating discussion. The help ofMr Robert Gilleon rat uterus. Arch Biochem 111:559

in revising the language is acknowledged Pearse A (1960): Freeze-drying and freeze-substmtution. In Pearse A. ed.
Histochemistry, theoretical and applied. London, Churchill, 25

Pearse A, PolakJ (1974): Bifunctional reagents as vapour- and liquid-phase

Literature Cited fmxatmves for immunohistochemistry. Proc R Microsc Soc 9:67

Agnati F, Fuxe K, Zu
Z, H#{228}rfstrand A, Okret 5, Wikstr#{228}m A, Coldstein Raarn 5, Nemeth E, Tamura H, O’Brien D, Cohen J (1982): Immuno-
M, Zoli M, Vale W, Gustafsson J (1985): Morphometrical analysis of the histochemical localization ofestrogen receptors in human mammary carci-
distribution of corticotrophin releasing factor, glucocortmcomd receptor and noma using antibodies to the receptor protein. Eur) Cancer Clin Oncol 18:1
phenylethanolamine-N-methyltransferase immunoreactive structures in the
Schneider W, Toft D, Sullivan W, Shyamala G (1988): Interaction of mu-
paraventricular hypothalamic nucleus of the rat. Neurosci Lett 54:147
rine progesterone receptors with specific monoclonal antibodies to the avian
Bayer E, Wilchek M (1980): The use ofthe avidmn-biotmn complex as a tool progesterone receptor. J Steroid Biochem 29:297
in molecular biology. In Glick D, ed. Methods of biochemical analysis. Vol
Sheridan P, BuchananJ, Anselmo V. Martin P (1981): Unbound progester-
26. New York, John Wiley & Sons, 1
one receptors are in equilibrium between the nucleus and cytoplasm in
Er#{228}nk#{228}
0 (1967): The practical histochemical demonstratmon of catechol- cells of the rat uterus. Endocrinology 108:1533
amines by formaldehyde-induced fluorescence. J R Microsc Soc 87:259
Sullivan W, Beito T, ProperJ, Krco C, Toft D (1986): Preparation of mono-
Er#{228}nk#{246}
0 (1964): Histochemical demonstration ofcatecholamines by fluores- clonal antibodies to the avian progesterone receptor. Endocrinology 119:1S49
cence induced by formaldehyde vapor. J 1-listochem Cytochem 12:487
Tuohimaa P, RenoirJ, Radanyi C, Mester), Joab I, Buchou T, Baulieu E
Falck B, Hillarp N, Thieme G, Torp A (1962): Fluorescence of catechol- (1984): Antibodies against highly purified B-subunit of the chick oviduct
amines and related compounds condensed with formaldehyde. J 1-listochem progesterone receptor. Biochem Biophys Res Commun 119:433
Cytochem 10:348 Welshons W, Krummel B, GorskiJ (1985): Nuclear localization of unoc-
Falck B, Owman C (1965): A detailed methodological descrmption of the cupied receptors for glucocorticoids, estrogens, and progesterone in GEl3
fluorescence method for the cellular demonstration of biogenic monoamines. cells. Endocrinology 117:2140
Acta Univ Lund [Section Ill 7:1
Ylikomi T, Isola), GascJ, Tuohimaa P (1987): Sexual maturation-associated
Gasc), Renoir), Radanyi C,Joab I, Tuohimaa P, Baulieu E (1984): Progester- and estrogen-induced progesterone receptor expression in the bursa of
one receptor in the chick oviduct: an immunohistochemical study with an- Fabricius. J Immunol 10:3174