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Use of the peroxidase-antiperoxidase technique for differential staining of multiple cell types in the rat pancreatic islet.

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THE ANATOMICAL RECORD 209:417-422 (1984)
Use of the Peroxidase-Antiperoxidase Technique
for Differential Staining of Multiple Cell Types
in the Rat Pancreatic Islet
Department ofdnatomy, School of Medicine, Oral Roberts University,
Tulsq OK 74171
Multiple staining of the endocrine cells of the pancreatic islet
was studied in tissue obtained from adult rats. After fixation in Bouin's fluid
and processing for light microscopy, the unlabeled peroxidase-antiperoxidase
(PAP)technique was performed. Successful staining procedures used variations
of the PAP technique with 3,3'-diaminobenzidine (DAB) and 4-chloro-1-naphthol (CN) as chromagens. The purpose of this study was to stain as many of
the four primary cell types (A-cells, B-cells, D-cells, PP-cells) as possible either
simultaneously or sequentially using photomicroscopy. At optimum antibody
titer, there was minimal nonspecific background staining which made it possible to differentiate cell types by intensity of the chromagen. Any two cell
types can be shown by using DAB with the first antibody and CN with the
second. To demonstrate three cell types simultaneously, three methods which
altered dilutions and chromagens were used. The first method consisted of
decreasing dilutions of primary antibody with DAB and CN as the chromagens. The second method involved repetitive DAB applications resulting in
three intensities of brown. The third method used a DAB immersion after the
second cell type was stained. This produced a color differential so the third cell
type could be distinguished with CN. To demonstrate the three cell types
sequentially, a masking technique was introduced with photomicroscopy. In
order to block the preceding complex, the previous cell type (demonstrated by
CN) was restained with DAB at a n increased dilution. The next cell type was
then stained with CN. These four methods were tried in attempts to stain four
cell types in the same tissue section.
Immunocytochemistry is a n invaluable tool
in elucidating cell types in various systems,
such as in the identification of cell types in
pancreatic islet tumors and other applications in the field of pathology (DeLellis et al.,
1979; Hutson et al., 1979; Mukai et al., 1982;
Polak and Van Noorden, 1983).It has become
increasingly desirable to demonstrate more
than one antigen in the same tissue section
to determine topographic localization and
distribution (Orci et al., 1976; El-Salhy et al.,
1982, 1983). The unlabeled peroxidase-antiperoxidase (PAP) technique (Sternberger,
1979)is one of the most sensitive and specific
of all immunocytochemical methods (Montero, 1981; Childs, 1982; Grzanna, 1982;
Vacca, 1982) and for this reason it is the best
0 1984 ALAN R. LISS, INC.
available technique to use for multiple
In studying the pancreatic islet, it would
be advantageous to- simultaneously stain the
four principle cell types with different chromagens. Due to the limited number of available insoluble chromagens, alternate methods must be developed to depict more than
two cell types in the same tissue section. It is
the purpose of this paper to describe some
extensions of the PAP technique that were
tried in a n effort to determine the optimum
conditions that would permit the simultaneous or sequential staining of two, three, and
four cell types of the pancreatic islet.
Received November 28. 1983; accepted March 2, 1984
Pancreatic tissue from adult Sprague-Dawley rats was processed for light microscopy
after fixation in Bouin's fluid. The unlabeled
PAP technique was performed on 5-pm sections with excess sheep-antirabbit IgG
(SARG) a t 1:250 and PAP complex a t 1:400
for 1-hour incubations. The secretory products of the four primary islet cell typesinsulin (Cambridge Labs), somatostatin-14
(Peninsula Labs), glucagon (Dako Labs), and
pancreatic polypeptide (R.E. Chance, Eli
Li1ly)-were used as the primary antibodies
in overnight incubations. The four primary
antibodies were diluted to the appropriate
concentration using a 1 O : l ratio of phosphate-buffered saline (pH 7.2) and 0.5% bovine serum albumin. Optimal dilutions
should be determined for each lot of antiserum (Petrusz, 1983). Dilutions of the primary antibodies vary in different steps of the
procedures depending on the sequence chosen and the desired result (Table 1).
The antisera were used in combination
with the insoluble chromagen, 3,3'-diaminobenzidine (DAB) and the soluble chromagen, 4-chloro-1-naphthol (CN), using a daylight filter to facilitate viewing of positive
CN reactions. For multiple staining, the PAP
technique was performed, consecutively altering the primary antibodies, their corresponding dilutions, and the sequence of the
For staining only two cell types, any two
primary antibodies may be used with DAB
as the first chromagen and CN as the second
(Joseph and Sternberger, 1979).For staining
more than two cell types, the first primary
antibody used must have a high specificity
at a high dilution to limit background
Four protocols were used for staining more
than two cells in the pancreatic islet. Three
of these four methods involved various combinations of the primary antibodies and the
chromagens in order to obtain simultaneous
demonstration of three or four cell types. The
fourth method employed the use of photomicroscopy to illustrate the staining of multiple
cell types in sequence on the same tissue
section. These four methods are outlined
Method 1
For the simultaneous demonstration of
three cell types, three primary antibodies
were used at decreasing dilutions with the
two chromagens DAB and CN. The first primary antibody was applied at a high dilution
with DAB as the chromagen. The slides were
then washed in phosphate-buffered saline
(PBS); a PBS wash was always used between
each application of the primary antibody regardless of the method. The next primary
antibody was applied at a decreased dilution
using DAB again. The third primary antibody was applied at a further decreased dilution in combination with CN. For the
simultaneous demonstration of four cell
types, four primary antibodies were used at
decreasing dilutions using DAB in the first
three stains followed by CN.
Method 2
A second procedure makes use of different
levels of binding between varying dilutions
of primary antibodies with one chromagen.
The sequence used was as follows: The first
primary antibody was applied a t a high dilution with DAB, followed by the second antibody a t a lower dilution with DAB, and by
a third antibody at a n even lower dilution
and DAB. This method was varied to include
four primary antibodies in a similar sequence with the fourth antibody being the
least dilute.
Method 3
The third method involves using DAB in
a n immersion technique. The first cell type
was stained with DAB using the primary
antibody a t a high dilution. The second cell
type was stained with CN at a lower dilution.
The slides were then immersed with agitation in DAB for 2-10 minutes. The third primary antibody was applied at a still lower
dilution and the process was repeated with
CN as the chromagen. A fourth primary antibody can be applied at a n even lower dilution following a 2-10-minute immersion in
DAB, and CN as the final chromagen.
Method 4
For the sequential staining of three andlor
four cell types, a masking technique was
used. This involved reapplying the previous
primary antibody but changing the chromagen used to stain it. The first cell type was
stained with DAB using a high primary antibody dilution. The second cell type was
stained using a lower dilution and CN. The
tissue was photographed a t this point. The
latter primary antibody was reapplied a t a
higher dilution and stained with DAB. The
third primary antibody was applied at a decreased dilution using CN as the chromagen.
The tissue was photographed again and the
process was repeated for a fourth primary
antibody. Sequential photomicrographs were
used to record color changes a t each application.
Other methods were used in a n effort to
improve multiple staining. Acidification
(HC1) and dimethylformamide (DMF) were
used in a n attempt to remove bound antibodies. Normal sheep serum (NSS) and methanolic H202 were tried to reduce background
staining. Controls were used a s specified by
Sternberger (1979).
(Figures 1-6 are color illustrations that appear in the Color Section elsewhere in this
issue. These figures are located on pp. 392393.)
DAB (glucagon) and CN (insulin), respectively, were then placed in a DAB solution
for either 2 or 7 minutes. The third primary
antibody was then stained with CN (somatostatin). As illustrated in this figure, the slides
were in the solution for 7 minutes, resulting
in a brown, blue, and purple. Antisomatostatin was used here at 1:10,000 since the
antiglucagon that started the staining sequence was not a high-titer antiserum. The
attempts to stain four cell types were unsatisfactory with this method due to poor color
In the masking procedure, the entire technique was performed again after the second
cell type was stained with CN (Fig. 6). The
same primary antibody is reapplied at a n
increased dilution due to the higher affinity
of DAB compared to CN and restained with
DAB. This procedure better differentiates
color but increases the number of times the
In regard to dual antigen staining, any two technique is performed on a given section.
cell types of the pancreatic islet can be dem- This results in increased background stainonstrated using the two chromagens DAB ing or a complete absence of color reaction.
and CN. In Figure 1the dilutions of antiglu- The sequential staining of four cell types was
cagon and antiinsulin are 1:lOO. Three cell of questionable value since, in some cases,
types cannot be obtained with concentrations positive identification was difficult to ascerthis high because of increased background tain while in others, no staining occurred. To
staining when the third antibody is applied. stain four cell types using sequential phoFigure 2 illustrates the decreased intensity tomicrographs, the technique must be perof staining with increasing dilutions of an- formed six times on a given section. This
tiinsulin and antisomatostatin, 1:4,000 and much treatment not only hinders staining,
1:15,000respectively. The dilutions we found but also adds the risk of losing the tissue
to be most effective for each primary anti- from the slides before all the sequences are
serum are summarized in Table 1.
In simultaneously staining three cell types,
After several attempts to use HC1, DMF,
the first method, which used decreasing di- NSS, and methanolic H202 to improve mullutions of primary antibody and two chrom- tiple staining, they were discontinued. When
agens, resulted in three different colors: a these procedures were used in dual antigen
brown-black DAB reaction product, a light staining, the results were unchanged. When
tan DAB product, and a blue CN product they were used during repetitive applica(Fig. 3). We obtained the best results by using tions of the PAP technique, the results were
antisomatostatin a t 1:15,000 as the first pri- poor because of tissue release from the slide,
mary antibody. Efforts to simultaneously possible mixing of CN and DAB, and reducstain four cell types using this method were tion in CN staining.
The second method, using repetitive DAB
applications with decreasing dilutions of priThe demonstration of two and three cell
mary antibody, produced three intensities of types was successfully performed using the
brown (Fig. 4). It was difficult to obtain four PAP technique under controlled procedures
intensities of one color using one chromagen with specific primary antibody dilutions. We
and consequently this method cannot be used concur with Sternberger and Joseph (1979)
for simultaneous demonstration of four cell that the best method for dual antigen staintypes.
ing is the use of insoluble brown DAB folThe third method using DAB immersion is lowed by soluble blue CN.
illustrated in Figure 5. In this procedure, the
The most appropriate factors for successful
slides with two cell types already stained by triple antigen staining are optimal dilutions
TABLE 1. Dilutions for staining the four principle cell types of the rut pancreatic islet
Significant background
staining, very dark core.
Some background staining,
dark granular positive cells.
Limited background staining,
lighter positive cells.
No background, light but distinguishable cells. Good for
masking technique (Method 4).
Background staining, very
dark positive cells with
core staining.
Some background and core
staining, positive cells
Minimal background and core
staining, positive cells can
be distinguished.
Very little overall staining.
Intense background and core
staining, very black positive
Decreased background, black
Core is less intense, brown
Core is lighter, positive
cells are easily visible.
Very little background, cells
Cells lighter but visible.
Cells extremely light.
Background and core too dark,
very dark cells.
Significantly better background, dark cells.
Cells beginning to get lighter,
some background staining.
Background has not changed,
cells are lighter.
Background the same, cells
extremelv light.
'Optimal dilutions for each cell type for various multiple staining procedures.
of primary antibody, adequate washings of
slides between steps, and freshly prepared
chromagen solutions with or without timed
DAB immersion and agitation. The sequential demonstration of three and possibly four
cell types can be shown by DAB masking,
photomicrographs, proper antibody titer, and
relatively pure antisera. Among the problems encountered is that of background
staining which will occur if primary antibody
concentrations are not optimal, adequate
washings are not performed (especially after
the application of primary antibody), thick
sections are used, and/or deparaffhizing is
not complete (Polak and Van Noorden, 1983).
These problems become more prevalent as
the number of times the technique is used on
a section increases. The causes of such problems may be any of the following: 1) reactions of the second- or third-sequence linking
antibody (SARG)with the first sequence PAP
or primary antibody; 2) reactions of second or
third primary antibody or PAP with preceding sequence SARG; and 3) reactions of CN
and DAB with the preceding sequence of
PAP. In multiple staining of three and/or four
cell types, limitations arose at the fourth application of the technique. At times, background staining was too intense to distinguish any cell types and/or showed no blue
reaction product. This may be due to a n increase in cross-reactivity and/or a decrease in
the number of available binding sites.
Exposures to HC1 and DMF were used by
other investigators in attempts to elute tissue-bound antibodies and thus improve antigen staining (Vandesande and Dierickx,
1975; Erlandsen et al., 1976; Nakane, 1968;
Grzanna, 1982; Polak and Van Noorden,
1983). These methods were tried in accordance with specifications between antibody
applications and results were either unchanged or poor. The lower pH can possibly
disturb the sensitive tissue and facilitate CN
and DAB mixing (Sternberger, personal communication). Multiple staining of three cell
types was obtained without the use of HC1
and DMF and consequently elution is not
necessary for dual or triple staining. This is
in agreement with results obtained in other
laboratories (Lechago et al., 1978; Joseph and
Sternberger, 1979; Sternberger and Joseph,
1979; Roth, 1982).Pretreatment of slides with
3% normal sheep serum and methanolic
H202 was tried to reduce nonspecific background staining and to block endogenous
peroxidase activity (Hsu et al., 1981). After
attempts to reduce background staining with
these methods, our results indicated that
there was either no effect or actually reduced
CN staining.
Although we did not eliminate all possible
dilutions and sequences, the successful staining of the four antigens may yet be possible
with the following: alternate blocking agent,
other insoluble chromagens, optimal dilutions, and/or pure antisera. Additionally,
with the development of monoclonal antibodies and increased efficiency in their production (Bosman, 1983; Ormanns and Schaffer,
1983) the staining of four cell types in the
pancreatic islet using immunocytochemistry
should be possible.
The authors wish to thank Dr. Ludwig
Sternberger for his helpful conversations in
suggesting alternate procedures and for his
encouragement to publish this work; and Mr.
Garv McCaulev for his professional help in
doing the colorprinting.
42 1
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isle, differential, pancreaticum, typed, rat, staining, multiple, use, antiperoxidase, techniques, cells, peroxidase
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