Research in Veterinary Science xxx (2012) xxxxxx

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Research in Veterinary Science

journal homepage: www.elsevier.com/locate/rvsc

A simple and rapid immunocytochemical technique for detection

of cytokeratin, vimentin, and S-100 protein in veterinary diagnostic cytology
Mariko Sawa a, Akira Yabuki a,, Noriaki Miyoshi b, Kou Arai c, Osamu Yamato a
a

Laboratory of Clinical Pathology, Department of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
Laboratory of Veterinary Pathology, Department of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
c
Kagoshima University Veterinary Teaching Hospital, Kagoshima University, Kagoshima, Japan
b

a r t i c l e

i n f o

Article history:
Received 28 October 2011
Accepted 25 March 2012
Available online xxxx
Keywords:
Cytokeratin
Cytology
Rapid immunocytochemistry
S-100 protein
Vimentin

a b s t r a c t
The objective of this study was to establish a simple and rapid immunocytochemical technique that can
be used in veterinary diagnostic cytology. Air-dried impression smears were collected from canine
tumors. Samples of epithelial tumors, mesenchymal tumors, and malignant peripheral nerve sheath
tumors and melanomas were used for detection of cytokeratin, vimentin, and S-100 protein, respectively.
The labeled streptavidinbiotin system was used in the present study. Optimal xation was determined
using standard immunocytochemical procedures, and acetone xation was found to be the most effective. Optimal concentrations of primary and secondary antibodies were determined at a preset 5-min
incubation. Omission of H2O2 treatment, shortening the time for blocking and labeled-streptavidin incubation, and simplifying washing did not decrease immunopositive intensities or enhance false-positive
reactions. The described rapid protocol requires approximately 45 min without the use of any special
equipment.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Immunocytochemistry (ICC) has been an indispensable technique for diagnostic cytology in human medicine over the last
20 years (Skoog and Tani, 2011). This advanced diagnostic technique has recently been used in veterinary cytology (Hinghaus
et al., 2007, 2008), and it provides additional information for cytomorphological criteria by determining the origin of anaplastic
tumor cells such as those originating from poorly differentiated
carcinomas, sarcomas, and melanomas; correct identication of
these cells can be impossible using routine cytomorphology. For
example, detection of the intermediate lament proteins (cytokeratin and vimentin) helps determine whether tumor cells have originated from epithelial or mesenchymal cells (Ramos-Vara et al.,
2010). Detection of S-100 protein may help to discriminate the
neurogenic or melanocytic tumors (Ramos-Vara et al., 2010).
In human medicine, ICC can be used not only to classify tumors
of unknown origin but also to detect therapeutic markers or infectious agents (Skoog and Tani, 2011). In patients with breast cancer,
intraoperative ICC examination of sentinel lymph nodes is performed using an ultra-rapid technique (Salem et al., 2002; Francz

Corresponding author. Address: Laboratory of Clinical Pathology, Department of

Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 8900065, Japan. Tel./fax: +81 99 285 3561.
E-mail address: yabu@agri.kagoshima-u.ac.jp (A. Yabuki).

et al., 2011). In certain neoplasms, ICC has shown some promise

in differentiating benign and malignant tumors; it was also useful
for preoperative evaluation (Ersoz et al., 2008; Ligato et al., 2008;
Raggio et al., 2010). ICC is considered a powerful tool that assists
cytomorphological diagnosis.
In veterinary medicine, ICC is not fully utilized in diagnostic
cytology, although most veterinary clinical pathologists know of
its advantage and are skilled in ICC. ICC is performed based on
the procedure of immunohistochemistry (IHC). However, the
IHC-based standard procedure may be inconvenient for most busy
clinical pathologists because it requires many steps and is time
consuming. Therefore, establishment of a simple and rapid ICC procedure is required for ICC to be widely utilized, not only in diagnostic cytology but also in clinical and clinicopathological research in
veterinary medicine.
The aim of the present study was to establish a simple and rapid
ICC procedure for detection of cytokeratin, vimentin, and S-100
protein in canine tumor samples by modifying the standard ICC
technique.

2. Materials and methods

2.1. Sample collections
Impression smears were prepared from surgical biopsy specimens that were obtained from dogs admitted to Kagoshima

0034-5288/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.rvsc.2012.03.013

Please cite this article in press as: Sawa, M., et al. A simple and rapid immunocytochemical technique for detection of cytokeratin, vimentin, and S-100
protein in veterinary diagnostic cytology. Res. Vet. Sci. (2012), http://dx.doi.org/10.1016/j.rvsc.2012.03.013

M. Sawa et al. / Research in Veterinary Science xxx (2012) xxxxxx

University Veterinary Teaching Hospital, Japan. All biopsy specimens were histopathologically diagnosed by a veterinary pathologist. Imprinted slides were thoroughly air-dried and stored at
30 C until use. Epithelial specimens, including mammary anaplastic carcinomas (n = 2), thyroid adenocarcinoma (n = 1), adenosquamous carcinoma (n = 1), perianal gland adenoma (n = 1), and
transitional cell carcinomas (n = 2) were collected and these specimens were used for detection of cytokeratin. Mesenchymal specimens were collected from chondrosarcoma (n = 1), osteosarcoma
(n = 1), hemangiosarcoma (n = 1), brosarcoma (n = 1), and myxosarcoma (n = 1), and these specimens were used for detecting
vimentin. Specimens from poorly pigmented melanoma (n = 1)
and malignant peripheral nerve sheath tumor (n = 1) were also collected and used for detecting S-100 protein.
2.2. Reagents
Immunocytochemistry was performed using a peroxidase-labeled streptavidinbiotin system (LSAB, DakoCytomation, Glostrup, Germany). Mouse monoclonal anti-cytokeratin (clone AE1/
AE3, DakoCytomation), mouse monoclonal anti-vimentin (clone
V9, Thermo Fisher Scientic, Fremont, CA, USA), and rabbit polyclonal anti-S-100 protein (cat. No. Z0311, DakoCytomation) were
used as primary antibodies. Biotin-labeled goat anti-mouse
immunoglobulin (IgG, Vector Laboratories, Burlingame, CA, USA)
and goat anti-rabbit IgG (Vector Laboratories) were used as secondary antibodies. All antibodies were diluted in 1% bovine serum
albumin (BSA)/10 mM phosphate-buffered saline (PBS). A 3% BSA/
PBS solution was used as blocking solution. Reactivity was determined using a 3,30 -diaminobenzidine (DAB) system (Merck,
Darmstadt, Germany), and Mayers hematoxylin was used for
counterstaining. For negative controls, non-immunized mouse
IgG (DakoCytomation) and non-immunized rabbit IgG (R&D Systems, Minneapolis, MN, USA) were used instead of primary
antibodies.
2.3. Determination of the optimum xative
Acetone (1 min, 4 C), 10% neutral buffered formalin (NBF;
15 min, room temperature), and 95% ethanol (15 min, room temperature) were used as trial xatives in the present study. The
detection of cytokeratin, vimentin, and S-100 protein was tested
in these 3 xatives using standard ICC procedures.
Standard ICC in our laboratories was as follows: (1) thoroughly
air-dry the sample with cold air from a drier, (2) wash in distilled
water, (3) treat with 3% H2O2 for 30 min, (4) wash in PBS, (5)
block for 60 min, (6) incubate overnight at 4 C with primary antibodies (1:200 dilution for cytokeratin and vimentin; 1:400 dilution for S-100 protein), (7) wash in PBS, (8) incubate for 30 min
with secondary antibodies (1:200 dilution), (9) wash in PBS, (10)
incubate for 15 min with LSAB (ready-to-use), (11) wash in PBS,
(12) react with DAB-H2O2 for 5 min, (13) stop the reaction by rinsing in cold distilled water, and (14) counterstain with Mayers
hematoxylin.

2.4. Modication for rapid ICC

After determining the optimal xative, the incubation time was
shortened and staining procedures were simplied. The following
incubations with antibodies and LSAB were performed at 37 C.
The incubation with antibodies was preset at 5 min, and dilutions
of primary antibodies were xed at 1:10, 1:20, 1:50, 1:100; and
1:200 for cytokeratin and vimentin and at 1:100, 1:200, and
1:400 for S-100 protein. Similarly, dilutions of secondary antibodies were xed at 1:150, 1:450, and 1:750 with 5-min incubation. If
the immunopositive signal in the best dilution was weaker than
that of the standard ICC, incubation time was additionally tested
at 10 and 15 min. The LSAB was a ready-to-use solution, and its
incubation time was set at 5, 10, and 15 min. The best dilutions
of antibodies and the best incubation time of LSAB were determined by comparing the intensity of immunopositive signals to
the standard procedure. Staining procedures were further shortened and simplied as follows: H2O2 treatment was omitted, and
the blocking time was shortened to 510 min. PBS washing was
performed using a direct stream from a washing bottle for approximately 10 s.
3. Results
3.1. Determination of the optimum xative
The intensities of immunoreactivity and background staining
from 3 xatives are summarized in Table 1. For detection of cytokeratin, clear and strong immunopositive signals were observed in
all xatives (Fig. 1AC). For detection of vimentin, strong immunopositive signals were observed in acetone xation (Fig. 1DF). For
detection of S-100 protein, moderate immunopositive signals were
observed in all xatives (Fig. 1GI). Background staining was relatively lower in acetone than in other xatives.
3.2. Dilution and incubation time of antibodies
The investigations described below were performed in acetone
xation. With the anti-cytokeratin and anti-vimentin antibodies,
dilutions of 1:10, 1:20, 1:50, 1:100, and 1:200 were tested at 5min incubation, and clear and intense immunopositive signals
were observed at 1:50 dilution in cytokeratin and at 1:100 in
vimentin. For anti-S-100 protein antibody, dilutions of 1:100,
1:200, and 1:400 were tested at 5 min. Subsequently, 10- and
15-min incubations were tested, and clear immunopositive signals
were observed at 1:100 dilution for 10-min incubation.
For the secondary antibody, dilutions of 1:150, 1:450, and 1:750
were tested at 5-min incubation, and clear immunopositive signals
were observed at 1:150 dilution. For LSAB, 5 min of incubation was
sufcient to detect the signals. Thus, optimal conditions of reagents were determined as follows: anti-cytokeratin, 1:50 dilution
and incubated for 5 min; anti-vimentin, 1:100 dilution and incubated for 5 min; anti-S-100 protein, 1:100 dilution and incubated
for 10 min; secondary antibody, 1:150 dilution and incubated for
5 min; and LSAB, ready-to-use and incubated for 5 min.

Table 1
Immunopositive signals and non-specic background staining for detection of cytokeratin, vimentin, and S-100 protein in the 3 different xatives.
Assessment

Immunoreactivity
Background staining

Acetone

10% NBF

95% Ethanol

Cytokeratin

Vimentin

S-100

Cytokeratin

Vimentin

S-100

Cytokeratin

Vimentin

S-100

3+
1+

3+
1+

2+
1+

3+
1+

1+
2+

2+
2+

3+
1+

1+
2+

2+
2+

NBF: neutral buffered formalin, 1+: weak, 2+: moderate, 3+ strong.

Please cite this article in press as: Sawa, M., et al. A simple and rapid immunocytochemical technique for detection of cytokeratin, vimentin, and S-100
protein in veterinary diagnostic cytology. Res. Vet. Sci. (2012), http://dx.doi.org/10.1016/j.rvsc.2012.03.013

M. Sawa et al. / Research in Veterinary Science xxx (2012) xxxxxx

Fig. 1. Standard immunocytochemistry for detection of cytokeratin, vimentin, and S-100 protein. (AC) cytokeratin, mammary anaplastic carcinoma. (DF) vimentin,
osteosarcoma. (GI) S-100 protein, malignant peripheral nerve sheath tumor. (A, D, and G) aceton xation. (B, E, and H) 10% neutral buffered formalin (NBF) xation. (C, F, and
I) 95% ethanol xation. Bars = 30 lm. Counterstain, Mayers hematoxylin.

Table 2
Procedure of rapid immunocytology for cytokeratin, vimentin, and S-100 protein.
1
2
3
4
5
6
7
8
9
10
11
12

Fixation in acetone for 1 minute at 4 C

Washing with PBSa
Blocking with 3% BSA/PBS for 510 min
Incubation with the primary antibodies for 5-10 min at 37 Cb
Washing with PBSa
Incubation with the biotin-labeled second antibodies for 5 min at 37 C
Washing with PBSa
Incubation with peroxidase-labeled streptavidin for 5 min at 37 C
Washing with PBSa
Incubate with DAB for 5 min
Termination of the reaction in cold distilled water
Counterstaining with Mayers hematoxyline

10 s with direct stream from a bottle.

5 min for anti-cytokeratin and anti-vimentin antibodies, and 10 min for anti-S100 protein antibody.
b

3.3. Further shortening and simplication of protocol

Omission of the H2O2 treatment, shortening of blocking time,
and a brief PBS washing did not enhance false-positive reactions.

3.4. Rapid ICC for cytokeratin, vimentin, and S-100 protein

The rapid ICC procedure established in the present study is
shown in Table 2. Intensities and specicities of immunosignals
for cytokeratin, vimentin, and S-100 protein obtained using this rapid protocol were sufcient compared to those of standard protocols (Fig. 2). In addition, damage to cellular morphology and the
degree of exfoliation of cells were apparently mild in rapid ICC
compared to those in standard ICC in most of the samples (Fig. 2).

4. Discussion
The superiority of acetone xation in ICC has been demonstrated in human lymph node cytology (Salem et al., 2003). In veterinary cytology, a recent report successfully demonstrated clear
immunopositive signals for vimentin, S-100 protein, and melan-A
in canine mesenchymal tumors using acetone xation (Hinghaus
et al., 2008). In the present study, the suitability of acetone xation
was demonstrated by a comparative analysis using 3 different xatives. Since exfoliation of cells from the slides and fragmentation
of cytoplasm during the staining process are disadvantages of acetone xation in ICC (Valli et al., 2009), short-time xation was used
in the present study. One-minute xation in acetone was sufcient
for the detection of cytokeratin, vimentin, and S-100 protein from
air-dried cytological samples.
In the present study, it was demonstrated that NBF and 95% ethanol can be used for the detection of cytokeratin and S-100 protein
but not vimentin. NBF is the most popular xative in IHC, and its
usefulness for ICC was pointed out in veterinary cytology (Valli
et al., 2009). Ninety-ve percent ethanol is a commonly used xative for ICC in human cytology (Shidham et al., 2000; Maeda et al.,
2005). However, the present study found no advantage between
NBF and 95% ethanol compared to acetone as a xative for the
detection of cytokeratin, vimentin, and S-100 protein.
Shortening of incubation time is a necessary step for accomplishing rapid ICC. Several methods for shortening the incubation
time of antibodies have been previously reported, and a very simple method has been used in a rapid intraoperative ICC in human
medicine (Maeda et al., 2005; Francz et al., 2011). Briey, this
method requires only highly concentrated antibody solutions,
and a few minutes of incubation is sufcient for detecting antigens
such as cytokeratin and cluster of differentiation (CD) antigens.
Although the present modication is based on this highly concen-

Please cite this article in press as: Sawa, M., et al. A simple and rapid immunocytochemical technique for detection of cytokeratin, vimentin, and S-100
protein in veterinary diagnostic cytology. Res. Vet. Sci. (2012), http://dx.doi.org/10.1016/j.rvsc.2012.03.013

M. Sawa et al. / Research in Veterinary Science xxx (2012) xxxxxx

Fig. 2. Comparison of rapid immunocytochemistry and standard immunocytochemistry. (A and B) cytokeratin, mammary anaplastic carcinoma. (C and D) cytokeratin,
thyroid adenocarcinoma. (E and F) vimentin, myxosarcoma. (G and H) vimentin, brosarcoma. (I and J) S-100 protein, poorly pigmented melanoma. (A, C, E, G, and I) rapid
immunocytochemistry. (B, D, F, H, and J) standard immunocytochemistry. Bars = 30 lm. Counterstain, Mayers hematoxylin.

trated antibody technique, non-economic consumption of antibody solution and the possibility of non-specic background staining were regarded as disadvantages of this simple method. Since
the present study emphasized easy handling, low background
staining, and observation of clear, specic immunopositive signals,
the qualities of cytokeratin, vimentin, and S-100 protein immunoreactivities were evaluated at different concentrations of antibodies for 5 min at 37 C. This incubation time and temperature
was determined on the basis of a previous report regarding the

ICC of human lymph nodes (Salem et al., 2003). Briey, this report
evaluated the immunoreactivity against cytokeratin using enhanced polymer one step (EPOS) method and demonstrated that
incubation with antibody for 5 min at 37 C resulted in acceptable
to strong immunoreactivity. In the present study, cytokeratin was
clearly detected in 1:50 (3.6 lg/ml) dilution of antibody, which
corresponded to the manufacturers recommended concentration.
Vimentin was clearly detected in 1:100 (2.0 lg/ml) dilution, which
corresponded to the lowest concentration recommended by the

Please cite this article in press as: Sawa, M., et al. A simple and rapid immunocytochemical technique for detection of cytokeratin, vimentin, and S-100
protein in veterinary diagnostic cytology. Res. Vet. Sci. (2012), http://dx.doi.org/10.1016/j.rvsc.2012.03.013

M. Sawa et al. / Research in Veterinary Science xxx (2012) xxxxxx

manufacturer (1:501:100; 2.04.0 lg/ml). The optimal concentration of secondary antibodies was determined to be 1:150
(10.0 lg/ml) dilution, which corresponded to the highest concentration recommended by the manufacturer (1:1501:750; 2.0
10.0 lg/ml). These ndings demonstrated that 5-min incubation
often accomplished rapid and sufcient antigenantibody reactions without excessively concentrated antibodies. On the other
hand, highly concentrated primary antibody was required to detect
acceptable signals for S-100 protein, and the 1:100 dilution
(10.25 lg/ml) used in the present study corresponds to 4 times
the dilution recommended by the manufacturer (1:400; 2.56 lg/
ml). In addition, the incubation time required to detect the acceptable immunoreactivity was 10 min. The anti-S-100 protein antibody used in the present study was a rabbit polyclonal antibody,
in contrast to the monoclonal antibodies against cytokeratin and
vimentin; therefore, it was suspected that a monoclonal antibody
might be most suitable for the present rapid ICC method.
LSAB was chosen as a detection system in the present immunocytology because of its reliability, wide application, and low cost.
Although ready-to-use solution was used in the present study, 5min incubation was enough to perform a sufcient biotin-streptavidin interaction.
H2O2 treatment for the removal of endogenous peroxidase
activity is often omitted during intraoperative rapid ICC in human
medicine (Katayama et al., 2004). In the present study, the effect of
omission of H2O2 treatment was evaluated, and no enhancement of
false-positive reactions was found after the omission. However, if
the samples contain many endogenous peroxidase-rich cells like
macrophages, H2O2 treatment might be required for accurate interpretation of the immunosignals. In such cases, a mild reagent (for
example 0.03% H2O2) would be preferable because cellular morphology in smear cytology may be damaged by 3% H2O2 treatment
(Key, 2006). Actually, in the present study, damage to the cellular
morphology and exfoliation of cells were observed in the standard
ICC using 3% H2O2 treatment.
In conclusion, the present study established a rapid ICC method
for air-dried samples by modifying the standard protocol. This rapid method enabled detection of cytokeratin, vimentin, and S-100
protein in canine tumor cells, and satisfactory immunopositive signals could be observed within 45 min. In this technique, simplicity
and easy handling are emphasized because no special equipment is
required to perform this rapid ICC. In small animal medicine, several antibodies are currently available for ICC (Ramos-Vara et al.,
2010), and the rapid ICC method established in the present study
may be used for many other antibodies. However, the type of
xative and the dilution and incubation time of the antibody

should be determined in advance. In addition, the tissue of origin

in the sample and type and degree of inammation should be
pre-checked to determine whether endogenous peroxidase blocking is necessary.

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Please cite this article in press as: Sawa, M., et al. A simple and rapid immunocytochemical technique for detection of cytokeratin, vimentin, and S-100
protein in veterinary diagnostic cytology. Res. Vet. Sci. (2012), http://dx.doi.org/10.1016/j.rvsc.2012.03.013