Comparison of Formalin- and Acetone-fixation
for Immunohistochemical Detection of
Carcinoembryonic Antigen (CEA) and Keratin
TOHRU KAKU, M.D., J. KENNETH EKEM, A.I.M.L.S., CONCHITA LINDAYEN, B.S., DENIS J. BAILEY, M.D.
A. W. PETER VAN NOSTRAND, M.D., AND EMMANUEL FARBER, M.D.
The purpose of the present study was to compare the relative
merits of cold acetone and buffered formalin as fixatives for the
detection of carcinoembryonic antigen and keratin in permanently
embedded tissues using a peroxidase-antiperoxidase (PAP) immunohistochemical procedure. The effect of treatment with the
proteolytic enzyme pronase also was examined in the formalinfixed tissues. Cold acetone was found to be superior to formalin
for the preservation of CEA and keratin antigenic activities in
a variety of benign and malignant tissues. Pronase treatment
markedly increased the staining intensities of both antigens in
formalin-fixed tissues. For many tissues, however, superior results were obtained using the cold acetone method, and this
technic is recommended for the optimum retention of antigenic
activity in permanently embedded tissues. (Key words: Fixation;
Cold acetone; Formalin; Immunohistochemistry; Pronase digestion; Carcinoembryonic antigen; Keratin) Am J Clin Pathol 1983;
80: 806-815
Department of Pathology, Toronto General Hospital
and University of Toronto, Toronto, Canada
may be preferable to these other methods, especially when
it is combined with embedding of the tissue in low-melting-point paraffin wax. 1516
In the present communication we compare the effects
of cold acetone fixation and formalin fixation for the
detection of carcinoembryonic antigen (CEA) and keratin
in sections from a variety of normal and malignant human
tissues. Sections from formalin-fixed tissues were examined both with and without pronase treatment. The
peroxidase-antiperoxidase (PAP) procedure was used to
localize antigens in tissue sections.
IN THE PAST FEW YEARS there have been several
remarkable advances in the methods used to localize specific antigens in histologic sections from formalin-fixed
tissues. One of the more important of these advances has
been the use of proteolytic enzymes, such as trypsin, pepsin,
and pronase, to "unmask" antigenic determinants that
otherwise are detected only inconsistently in routinely processed sections. This procedure has been shown to increase
both the reliability and sensitivity of immunofluorescence
and immunoperoxidase methods.4"8,11,12,14'18"20,27
In spite of the apparent contribution of proteolytic
enzymes, it is not unlikely that formalin fixation results
in an irreversible reduction or even abolition of certain
antigenic determinants. Until quite recently the only acceptable alternatives to formalin fixation were the use of
fresh frozen or cold ethanol-fixed tissues.3,7,21 However,
recent studies have suggested that fixation in cold acetone
Materials and Methods
Received November 29, 1982; received revised manuscript and accepted for publication June 2, 1983.
The authors wish to acknowledge the generous support of the Charlie
Conacher Research Fund and the Queen Elizabeth Hospital Research
Foundation.
Address reprint requests to Dr. Farber: Department of Pathology,
University of Toronto, 100 College Street, Toronto, Ontario, Canada
M5G 1L5.
A variety of surgically resected human tissues was received from the Surgical Pathology Service of the Toronto
General Hospital. Two contiguous tissue blocks, each
approximately 2-3 mm thick, were removed from these
specimens within one hour of receipt of the tissues in the
laboratory.
For the CEA study, 27 tumors were examined, including 17 colonic adenocarcinomas, two colonic adenomas, four lung adenocarcinomas, two gastric adenocarcinomas, and two squamous cell carcinomas of lung.
For the keratin study, eight tumors were examined,
including five adenocarcinomas (two from stomach, two
from breast, and one from lung) and three squamous cell
carcinomas (one from esophagus, one from skin, and one
from lip). Also included were normal specimens of skin,
breast, and salivary gland.
One of the contiguous tissue blocks was processed by
cold acetone fixation and was embedded in low-meltingpoint paraffin wax (Table 1, A). The other was fixed in
10% neutral buffered formalin for 24 hours at room temperature. Further processing of the formalin-fixed tissue
was carried out in a routine histopathology laboratory.
0002-9173/83/1200/0806 $01.30 © American Society of Clinical Pathologists
806
Vol. 80 • No. 6
FIXATION AND IMMUNOHISTOCHEMISTRY
Serial sections were cut at 6 ^m and were mounted
on clear glass slides. When enzyme treatment was to be
performed, the slides were precoated with LePage Bond
Fast white glue.''
Following deparaffinization and rehydration, selected
sections from each of the formalin-fixed tissues were subjected to proteolytic enzyme treatment. Solutions of pronase (protease type VII, Sigma) were prepared immediately before use at concentrations of 0.05% and 0.025%
in 0.05 M Tris-HCl buffer, pH 7.6.14 The enzyme solutions
and the sections were allowed to equilibrate at 37°C, and
then enzyme treatment was carried out at this temperature
for either 10 or 15 minutes. Sections that were not exposed
to pronase were immersed in cold phosphate-buffered
saline (PBS) for an equivalent length of time. Following
enzyme treatment, the sections were washed thoroughly
in cold PBS to remove residual enzyme before the immunostaining technic was carried out.
CEA and keratin were detected in tissue sections by
means of the PAP method. 24 The different steps in this
procedure are listed in Table 1, B. In some cases sections
were treated with 0.3% hydrogen peroxide in absolute
methanol immediately following incubation in graded
alcohols, in order to inhibit endogenous peroxidase activity. Generally, however, the tissues that were examined
in this study showed negligible amounts of endogenous
peroxidase activity, and, consequently, this step usually
was omitted. Control sections were incubated with normal
rabbit serum in place of specific antiserum. Additional
sections were stained with hematoxylin and eosin for routine histologic examination. Anti-CEA antiserum, swine
antirabbit IgG antiserum, horseradish peroxidase-rabbit
antihorseradish peroxidase complex (PAP), normal rabbit
serum, and normal swine serum were purchased from
Dakopatts A/S of Copenhagen, Denmark. Keratin (from
human callus) and an antikeratin antiserum were prepared
by the methods of Sun and Green. 25
Before use the anti-CEA antiserum was absorbed with
a perchloric acid extract of spleen in order to remove
antibodies that might react with nonspecific cross-reacting
antigen. Portions of three normal human spleens obtained
at autopsy were extracted with 1.2 N perchloric acid by
the method of Primus and associates.17 The centrifuged
and lyophilized extract was mixed with the antiserum,
in a ratio of 100 mg of extract per 1.0 mL of antiserum,
by continuous rotation for 1 hour at 37°C and for 18
hours at 4°C. The fine precipitate that formed was removed by centrifugation at 20,000 g for 30 minutes, and
the activity of the absorbed antiserum was assessed by
double immunodiffusion in agar gel against the spleen
extract used for absorption and against a similar extract
prepared from a colonic adenocarcinoma obtained at surgery. The absorbed antiserum showed no reactivity against
the spleen extract and showed only a single, intense pre-
807
Table 1. Low-Melting-Point Paraffin Method
for Immunohistochemistry
A. Preparation of Tissue
1. Fix tissue blocks (2-3-mm thick) in cold acetone (4°C) for 1424 hours.
2. Dehydrate with two changes of fresh cold acetone—30
minutes for each change.
3. Clear in benzene (or xylene) at room temperature—three
changes for 15 minutes each.
4. Transfer into benzene (or xylene)—low-melting-point paraffin
wax* mixture (1:1 v/v) and maintain at 49°C for 15 min.
5. Place in low-melting-point paraffin wax at 49°C for 60
minutes. Replace with fresh wax two times over this period
(at 20-minute intervals).
6. Embed in standard paraffin wax (paraplast).
7. Cut 6-M sections onto clean glass slides and air dry. The
floatation medium should be maintained at 44°C.
B. PAP Method
1. Deparaffinize sections with two changes of xylene, and then
hydrate through graded alcohol solutions to distilled water.
2. Place sections in cold phosphate-buffered saline (PBS) pH 7.1
for 10 minutes. (Enzyme treatment with pronase is carried
out following this step. See text for details).
3. Wash in three changes of cold PBS for five minutes.
4. Apply nonimmune swine serum diluted 1:10 in PBS (v/v) for
10 minutes at room temperature.
5. Drain off excess serum (do not rinse).
6. Apply primary rabbit antiserum of appropriate dilutionf (anti
CEA 1:400-1:3,200, antikeratin 1:100-1:3,200). Incubate
overnight at 4°C.
7. Wash in three changes of cold PBS, each for 5 minutes.
8. Apply swine antirabbit IgG serum 1:10 dilutionf for 30
minutes at room temperature.
9. Wash in three changes of cold PBS, each for 5 minutes.
10. Apply rabbit peroxidase-antiperoxidase complex (PAP) at a
1:50 dilutionf for 30 minutes at room temperature.
11. Wash in three changes of cold PBS, each for 5 minutes.
12. Stain for 2 minutes at room temperature in freshly prepared
DAB solution:
0.05 M Tris-HCl buffer, pH 7.6
3,3'-diaminobenzidine tetrahydrochloride
13.
14.
15.
16.
100 mL
50 mg
Dissolve, filter, and just before use add 1 mL 1% hydrogen
peroxide in distilled water.
Wash thoroughly in running tap water for several minutes.
Counterstain with Carazzi's haematoxylin for 2 minutes.
Wash in running tap water for 5 minutes.
Dehydrate through graded alcohols, clear in xylene, and
mount in Permount.
• BDH Chemicals.
t Dilutions are made in PBS containing 1% normal swine senim.
cipitin band when reacted against the colonic adenocarcinoma extract.
Results
The staining reactivity of CEA appeared to be best
preserved in tissues processed by the cold acetone method
(Figs. 1 and 2, Table 2, A). However, in formalin-fixed
tissues the staining reaction of CEA was improved significantly by pronase treatment (Figs. \A, \B, 1A, and
2B, Table 2, A). In sections prepared from cold-acetone-
KAKU ET AL.
808
A.J.C.P. • December 1983
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FIXATION AND
Vol. 80 • No. 6
IMMUNOHISTOCHEMISTRY
809
FlG. 1. Immunohistochemical localization of CEA in sections of moderately differentiated adenocarcinoma of colon and adjacent histologically
normal mucosa. A (upper). Formalin-fixed tissue. B (lower). Formalin-fixed tissue, section pretreated with pronase. In each photomicrograph the
adenocarcinoma is toward the left and the histologically normal mucosa is toward the right. In the formalin-fixed tissue, pronase treatment enhances
the staining reaction for CEA in the cytoplasm and at the luminal aspect of the adenocarcinoma cells. The histologically normal mucosa shows a
positive staining reaction in the cold-acetone-fixed tissue, (see Fig. \C) but in the formalin-fixed tissue the reaction is positive only in the pronasetreated section. Anti-CEA antiserum dilution 1:1,600. Counterstained with hematoxylin (XI25).
fixed tissues and in pronase-treated sections from formalin-fixed tissues, all 17 colonic adenocarcinomas and
the two colonic adenomas showed clearly positive CEA
staining reactions. On the other hand, when pronase
treatment was omitted, the sections from the formalinfixed tissues showed only weak or very weak staining
reactions in 9 out of 17 adenocarcinomas. In all of the
colonic adenocarcinoma specimens examined, the histologically normal mucosa bordering the carcinoma exhibited a positive staining reaction for CEA when acetonefixed and formalin-fixed, pronase-treated sections were
used. However, when formalin-fixed, nonpronase-treated
sections were used, the normal mucosa showed only very
weak staining for CEA infiveout often cases and negative
staining for CEA in three out often cases (Fig. 1). In 10
out of 12 cases of colonic adenocarcinoma, the histolog-
ically normal mucosa that was remote from the tumor
(greater than 2 cm) showed moderately or strongly positive
reactions for CEA in cold-acetone-fixed and formalinfixed, pronase-treated sections. In these same cases, however, formalin-fixed sections that were not treated with
pronase showed only weak or very weak staining reactions.
Therefore, the staining reaction for CEA was improved
significantly in both normal and neoplastic tissues by
pronase treatment (Figs. IB and IB, Table 2, A).
Keratin also appeared to be best preserved in tissues
that were prepared by the cold acetone method (Fig. 3B).
When keratin antiserum was used at a dilution of 1:1,000,
formalin-fixed sections invariably showed less intense
staining than was evident in acetone-fixed sections from
the same specimens (Fig. 3). Pronase treatment increased
the staining intensity of keratin in formalin-fixed tissues,
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IMMUNOHISTOCHEMISTRY
811
FlG. 2. lmmunohistochemical localization of CEA in sections of well-differentiated adenocarcinoma of lung. A (upper). Formalin-fixed tissue. B
(lower). Formalin-fixed tissue, section pretreated with pronase. In the formalin-fixed tissue, pronase treatment enhances the staining reaction for
CEA. The staining reaction in the cold-acetone-fixed tissue (see Fig. 2C) is at least equivalent to that in the pronase-treated section. The hematoxylin
and eosin stained section (see Fig. 2D) shows that the cold acetone method results in preservation of morphologic detail comparable to that obtained
in formalin-fixed tissues. Anti-CEA antiserum dilution 1:2,600 (XI25).
<
but even following this treatment the reaction was never
as intense as in cold-acetone-fixed materials (Fig. 3, Table
2, B). It was an interesting observation that, in addition
to positive staining for keratin in normal epidermis and
in all of the squamous cell carcinomas examined, five of
the adenocarcinomas tested and normal tissue from breast
and salivary gland also showed positive staining reactions.
Other investigators also have reported that adenocarcinomas that show negative staining for keratin in formalinfixed tissues22 may show positive staining reactions when
alcohol-fixed or frozen sections are used.1
Overall, specimens processed by the cold acetone
method showed more intense staining for CEA and keratin
than the same specimens processed by the formalin fixation method (Table 2). With both methods the staining
intensities of the antigens remained unaltered when tissue
blocks were stored for periods of up to one year at ambient
room temperatures. In formalin-fixed tissues, the staining
intensities of the antigens invariably were improved if
the sections were treated first with pronase. The optimum
conditions for pronase treatment were found to be a
10-minute incubation at 37°C, using the enzyme at a
concentration of 0.025% w/v in 0.05 M Tris-HCl buffer,
pH 7.6. Longer periods of incubation or higher concentrations of the enzyme were either deleterious to the sections or had no effect on the ultimate staining reaction.
Pronase treatment did not appear to reverse completely
the antigen-masking effect of formalin fixation (Figs. 2B
and 3A, and Table 2).
No staining of any tissue for CEA or keratin was detected when specific antisera were replaced with normal
rabbit serum.
Discussion
Neutral buffered formalin is the most common fixative
used in North America for the routine processing of surgically resected tissues. For this reason it is an appropriate
fixative upon which to base evaluations of the effectiveness
of alternative fixatives for such technics as immunohistochemistry. Comparisons based upon neutral buffered
formalin as a standard have the additional advantage of
providing an indication of the usefulness of the large
amounts of tissue that already have been processed in
this fixative and are maintained in paraffin-embedded
blocks.
The results of the present study clearly show that fixation in cold acetone affords a higher level of sensitivity
for the detection of CEA and keratin in a variety of normal
and neoplastic tissues than does fixation in neutral buffered formalin. Furthermore, this difference in sensitivity
persists for at least one year following fixation and
embedding, which suggests that the cold acetone fixation
method is compatible with the long-term stability of these
antigens. If sections from formalin-fixed tissues are treated
with pronase prior to immunohistochemical staining, the
results that are obtained closely approach those obtained
using the acetone fixation method. The increased efficacy
of immunohistochemical staining following pronase
treatment is similar to that noted by Huang and co-workers" using pretreatment with trypsin to detect a variety
of protein antigens (including CEA) by immunofluorescence methods. Pronase treatment appears to be preferable
to treatment with trypsin because it involves a considerably shorter period of incubation and because at optimal
concentrations it produces less damage to tissue sections.
The use of the PAP method to localize antigens in
formalin-fixed tissues has opened a wide variety of applications of histochemistry to diagnostic pathology.
However, the method is not entirely reliable unless sections are pretreated with proteolytic enzymes. 4,5,81418 The
present study confirms that pronase treatment greatly
facilitates the localization of antigens in formalin-fixed
tissues. This procedure no doubt will continue to be useful
in retrospective studies of formalin-fixed tissues already
stored in paraffin blocks.
An important conclusion from this study is that the
results of immunohistochemistry from one laboratory to
another are not always comparable unless similar technical procedures have been used. In interpreting the results
from different laboratories, careful note must be made
of such variables as the conditions and duration of fixation, embedding procedures, and in particular, whether
or not special technics, such as proteolytic enzyme treatment, have been used to enhance the sensitivity of immunohistochemical staining. These considerations are
especially important when the presence or absence of a
specific antigen is used as the basis for diagnosis in anatomic pathology.
It also should be pointed out that enhanced sensitivity
always may not be a desirable characteristic in an immunohistochemical procedure. For example, the observation that keratin is detected more easily in squamous
cell carcinomas than in adenocarcinomas suggests that a
method with relatively low sensitivity for this antigen,
FIG. 2. C (upper). Cold-acetone-fixed tissue. D (lower). Cold-acetone-fixed tissue,
section stained with hematoxylin and eosin (no immunochemical staining).
Vol. 80 • No. 6
FIXATION AND IMMUNOHISTOCHEMISTRY
813
FIG. 3. Immunohistochemical localization of keratin in sections
of poorly differentiated adenocarcinoma of stomach. A (upper, left).
Formalin-fixed tissue, section pretreated with pronase. B (upper, right).
Cold-acetone-fixed tissue. C (lower, left). Cold-acetone-fixed tissue,
section stained with hematoxylin and eosin (no immunochemical
staining). The staining reaction in the cold-acetone-fixed tissue is
more intense and more sharply defined than in the pronase-treated
section of the formalin-fixed tissue. Keratin was undetectable in the
formalin-fixed tissue if pronase treatment was omitted (not shown).
Antikeratin antiserum dilution 1:1,000 (XI25).
814
A.J.C.P. • December 1983
KAKU ET AL.
Table 2. Relative Staining Intensities* of CEA and Keratin in Formalin-fixed
and Cold-acetone-fixed Tissue Sections
B. Iterating
Primary Antiserum Dilution
A. CEAf
Primary Antiserum Dilution
Section
1:400
1:800
1:1600
1:3200
1:100
1:200
1:400
1:800
Formalin-fixed, no pretreatment
Formalin-fixed, pronase pretreatment§
Cold-acetone-fixed
++
+++
+++
+
+++
+++
+
+++
+++
±
+
+
+++
+++
+++
++
+++
+++
+
+++
+++
++
+++
• + + + = strong; + + • moderate: + " weak; ± = marginally detectable; - = negative.
t Sections of moderately differentiated adenocarcinoma of colon were used to assess staining
of CEA.
such as assessment in formalin-fixed, nonenzyme-treated
sections, may be a useful procedure for distinguishing
these two forms of malignancy. Other examples where
reduced sensitivity might be diagnostically helpful are in
distinguishing adenomas from adenocarcinomas in colonic biopsies (based on the higher CEA content of the
latter10'23) and in identifying transitional cell carcinomas
of bladder that show a propensity to recur or become
invasive (based on observations that there is a correlation
between these properties and low or absent blood group
antigens on the neoplastic cells913).
The results of the present study indicate that acetone
fixation combined with embedding in low-melting-point
paraffin should be considered as an alternative to formalin
fixation in prospective immunohistochemical studies.
This approach readily permits localization of antigens
that do not survive the rigors of routine fixation and
embedding. One other advantage of the cold acetone fixation method is that it is compatible with the histochemical analysis of several enzyme activities, including acid
phosphatase, alkaline phosphatase, nonspecific esterase,
cholinesterase, gamma-glutamyl transferase, and aminopeptidase.215,26 Therefore, it provides an opportunity
to correlate the localization of these and other enzyme
activities with the localization of antigenic components
by immunohistochemical methods. Such correlations may
prove useful in the analysis of a wide variety of normal
and neoplastic human tissues.
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1:3200
+
++
±
+
+
} Sections of normal skin were used to assess staining of keratin.
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