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© © All Rights Reserved
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/ 39
Gea 5 oo a
— Handbook of
mea iuicecte (oes
Staining Methods
is Janice A. Bourne
Pa
ate
ee Sa QV Ve
Ge 4:
BAA
R OMe ‘iver section from a hepatoblastoma case stains positively for
the oncoletal antigen alpha-tfeoprotein (AFP). A. major
lyeonrotein ofthe fetus, AFP has bean localized in hepatocel
Tar eareinama and yok sac tumors ol the ovary and tests. AFP
's 2 useful marker far astingulsring these types of tumors from
other neoplasms
Skin, $100
‘A skin biopsy ftom a case of superficial, spreading, malignant
‘meianoma showing intense postive staining for §-100 prota.
S100 is & nervous system ass
‘marker for the entication of melanocyte derived tumors such
fas nevi and melanoma, regardless of melanin content. It can
falso aid n distinguishing poorly differentiated melanoma from
tumors of ebscure histological origin.
Skin, Keratin
‘sin Biopsy from a patient with well fers
Call carcinoma shows pastvesiaining for keratin. The variation
In-sain intensity Is due tothe diference inthe amount of keratin
produced by the indivigual tumor cel. Keratin Is a useful
‘marker Tor establishing the epithelial nature of primary and
‘metastaie tumors, especialy for thase exhibiting uncharacter
'stie morphology
AO a
Skin, Papillomavirus.
Immunoparonidase staining for papilomavius shows lca!
[zation in tre nucel of infected cells present in his skin biopsy
Papliomavius can be loaned sn’ variety of proliferative
squamous lesions as well as in many cases of cervical
‘dysplasia. Some lypes of papillomavirus have the abilly 10
Undergo. malignant ansformation especialy in immunosup
Dressed patients, Incieations of papillomavirus infection have
‘boon found Ina high percentage of women wilh cervical
necplasia.
Liver, HBsAg
Hepa B surlace antigen (HBsAg) representing he capsular
‘material of the hepaiis B vius can be localized i the
fytoplasm of infected liver cells. HBeAg can be detected in
‘cases of type B viral hepatiis, chronic carers, cfrhosis, and
Fepatoceliar carcinoma. The results of immunoperoxidase
Staining are usvally more intense and easier to interpret than
those achieved by using an orcein sain.
Colon, PSA
Positive slaining for prostate specific antigen (PSA) icentites
this colon tumor a6 being metastatic from the prostate. All
primary and metastale prostatic carcinomas show positive
Staining for PSA regardless of tne morphological differentia:
tion, hile nonprostalle malignancies do not sain PSA Is a
Useful tool n the isentfication oF tumors of prostatic origin,
‘The formalin tied, parattin embedded tisue specimens usod
{or inese photographs ware all slained using the peroxidase:
antiperoxiGase (PAP) technique. The chromagen, Samino3:
‘ethylearbazole, produces @ red end product that preciptates at
the site of the antibody reaction. Positive cals wll tnerofore
Stain red hile negative cols and nucle will appear blue due to
the hematoxylin counterstain.
"Note adaltionat photographs on inside back cover. Handbook of
Immunoperoxidase
Staining Methods
Janice A. Bourne
Immunochemistry Laboratory
DAKO CORPORATION ‘Acknowledgements
‘Special thanks are extended to Thomas Boenisch, Laboratory Director and Therese McCann, Research Technologist, both of
DAKO CORPORATION: for their technical input and editorial suggestions.
Kay Velez did a wonderful ob translating my handwritten manuscrit into readable typewriting, Al of my coworkers at DAKO
CORPORATION helped by sharing the routine workload so | could see this project through, and by providing moral support when it
was needed
Lastly, am grateful to Viggo Harboe, President at DAKO CORPORATION, for the opportunity to write this book with a free
hand; and to David MeCarthy for his advice and help in its preparation and production.
‘Copyright © 1983 by DAKO CORPORATION. All rights reserved. Printed in the United States of America
DAKO CORPORATION, 22 North Milpas Street, Santa Barbara, CA 93103, TABLE OF CONTENTS
‘Antigens and Antibodies
Antibody structure; preduction; types of antibody solutions; monoclonal antibodies.
Staining Methods
Peroxidase: direct, indirect, PAP and avidin-biotin methods; endogenous peroxidase activity; nonspecific background
staining
Staining Procedures
Direct, indirect, PAP and avidin-biotin techniques; dilution methodology; butters, substrates; counterstaining, timing:
enzyme digestion,
Fixation and Processing
Methodology; adhesives; processing of smears, frozen and plastic embedded sections.
Controls
Types of controls used in immunoperoxidase staining,
Interpretation
Staining characteristics; effects of issue processing: artifacts.
‘Troubleshooting
How to find and solve staining problems quickly.
Limited list of pertinent references.
Index
4
25
29
at
32
36 Handbook of
INTRODUCTION
\With the introduction of immunochemical techniques into
the routine histology laboratory, a new era of tissue staining
evolved. These very sensitive and specific methods, utilizing
antigen-antibody complexes, allow visualization of previously
Undetectable cell components, This handbook will guide you
through the maze of scientific knowledge necessary to per-
form immunoperoxidase staining techniques.
‘We will star at the beginning by explaining exactly what
immunochericals are, and what they can do. Once we have
‘an understanding of the various methods that are used, we
‘can then outline stepby:step procedures; discuss controts,
fixation and processing of specimens; and provide special
hints to achieve successful staining, An entire chapter is
{devoted to troubleshooting, for those spacial problems. A sec:
tion on stain interpretation and reference list will ald in the
evaluation of the finished product
Immunoperoxidase staining is an important and useful tool
in the identification of a wide variety of cell products. This
handbook contains ail he necessary information fo assist you
in making these techniques an integral part of your laboratory
routine, Immunoperoxidase Staning Methods
——
ANTIGENS AND ANTIBODIES
Ils necessary to have a basic knowiedge of the building
blocks of immunology (antigens and antibodies) to more fully
understand immunoperoxidase methods.
Antigens
Antigens have two main properties. The first is immuno-
‘genicity, which is the ablity to induce antibody formation. The
‘second property Is specific reactviy, which means that the
‘antigen can react with the antioody it caused to be produced.
‘The reaction between an antigen and its antibody is one of the
most specific in biology, and is the reason that immunohisto-
chemical reactions are more precise than ordinary histo:
chemical techniques,
‘An antigen then, is a substance foreign to the host which
stimulates formation of a specific antibody and which will
react with the antibody produced. This reaction involves the
formation of immune complexes comprised of several an:
tigen and antibody molecules. These complexes may become
very large and form precipiiates which can be measured by
various techniques.
Antibodies
{An antibody is a serum protein that is formed in response
toexposure to an antigen, and reacts specifically with that an-
tigen fo form immune complexes either in the body or in the
laboratory. Antibody production is a response by the body to
foreign material (an antigen), and is designed to rid the body
ofthis invader.
Antibodies are contained in the gamma globulin raction of
serum, and are atten called immunoglobulins (Ig). They can
‘be divided into five classes based on their size, weight, struc
ture, function, and other criteria, The classes are IgA (im:
munoglobulin A), IgD, IgE, 1G, and IgM. Antibody solutions
utilized in immunohistochemical staining contain mostly 1G
type antibodies, with lesser amounts of the other classes
Antibody Structure
‘Structurally, an antibody is made up of two kinds of protein
chains—heavy and light chains. Immunoglobulins are named
for their heavy chains, 60 the IgG molecule in Figure 1 will
have heavy chains of the gamma { v) type. An IgA antibody
has alpha (« ) heavy chains; gD, delta 4 heavy chains; gE,
cepsiion (« ) heavy chains; and IgM has mu (j.) heavy chains.
‘A primary antibody for immunoperoxidase staining that is
“specific for gamma chains" wil localize the heavy chain of
fan IgG molecule.
‘There are only two types of light chains common to all ve
‘groups: kappa {x ) and lambda (4), An IgG molecule has two
Identical light chains, elther two kappa chains or two lambda
chains (Figure 2), A single antibody can never have both kap-
pa and lamoda chains. This Is important when discussing the
interpretation of light chain staining in lymphoma cases.
‘The orientation of an IgG antibody is shown in Figure 3.
The Fe portion stands for fragment crystalline, and will
crystallize out Upon purification. This region is involved in
‘complement fixation and transfer of antibody across the
placenta. The remaining portions are called the fragment an-
tigen binging or Fab regions. These are the parts of the an-
tibody molecule capable of specifically binding to the antigen,
This IgG molecule has the ability to bind two antigen mole-
cules, one at each Fab site.
Antibody Production
In order to produce an antibody for laboratory use, itis ist
necessary to purty an antigen. A source for the antigen such
‘as serum, urine or tissue is subjected to a combination of pro-
‘cedures including precipitation, centrifugation, dialysis,
chromatography and electrophoresis to obtain a highly
purified antigen, The antigen Is then injected into an animal of
different species than that of antigen source, The animal will
identity the antigen as foreign matter, and produce an an-
tibody directed specifically against it. Antibody production
begins within twenty minutes after injection, although
measurable quantity of antibody cannot be detected for 5-10
days. Small blood samples are usually obtained and pooied at
two week intervals. Booster injections of antigen are often
‘administered every month to promote consistent antibody
production
“The choice of an animal for injection depends upon the an
tigen used, housing faclties available, amount of antibody
needed, and personal preference. Usually several animals of
the species chosen will be injacted with an antigen, as each
animal will vary in how it responds to the antigen, and in the:
‘amount of antibody it produces. After several bleedings are
pooled, contaminants present must be removed. This is
usually accomplished by elther liquid or sold phase antigen
‘absorption techniques. 8 Hancbook of
Heavy Chain Heavy Chain Light chain
(amme) (gamma) ‘happs)
Light Chain ght chain Light chain
lambda)
INA
Figure 1. lgG molecule showing paired hoavy chains of gamma
type.
Figure 2 I molecules showing only possible ight chain
configurations.
Fe
Fab Fab
a a
A
f *, Bad,
a
ayn eh ee
a a 4
Figure 3, Dision of IgG molecule ito Fe and FAb fragments.
‘Antigen binding occurs a he two FAD sites.
Figure 4. Whole serum antibady solution containing al normal
‘animal serum companents in adlion to speciic antibody.
K-44
RX
Figure . immunoglobulin fraction of serum containing only
aniibedies
Figure 6 Antigen specific antibody is more speci than usualy
K
RX
ex
9
Figure 7. conjugate combines an antibody and some type of
visual marker.
Key to Figures 4 through 8
Jlseancy 9h com nan
as
Figure 8, A peroxiase-aniperoxidase immune complex formed
bynatural antes batween antigens and antibodies.
© visvarnercer — MABE serum components
© Peroxidase Enzyme Immunoperoxidase Staining Methods
‘Types of Antibody Solutions
‘There are several antibody preparations available for use
In Immunoperoxidase procedures, The easiest to produce,
land therefore the most common and least expensive, is,
‘whole serum. Animal blocd containing the antibody is cen-
trifuged to separate the cells from the serum, and any con-
taminating antibodies are absorbed out.
‘A whole serum solution is pictured schematically n Figure
4, It will contain antibodies specific forthe antigen the animal
‘was immunized with, Other antibodies which are products of
‘the animal's normaly functioning immune system will also be
‘resent, These should not interfere with staining procedures.
‘The bulk of the whole serum fraction Is made up of ordinary
serum components such as enzymes, electrolytes, and
‘serum proteins. Occasionally, these other serum elements,
‘can cause unwanted background staining in some tech-
niques. This is due to the affinity of serum proteins, most
notably albumin, alpha and beta globulins, for certain tissue
components,
Since the only element necessary for immunoperoxidase
methods is antibody, all other serum components can be
eliminated. This type of preparation, called an immuno-
Globulin oF Ig fraction, is depicted in Figure 5. This solution
contains mostly antibodies, both specific and naturally occur.
ring, plus @ very small amount of residual serum protein. The
removal of the majority of proteins will reduce the chances of
‘nonspecific reactions in various techniques.
It is possiple, as shawn in Figure 6, to prepare a solution
containing oniy antibodies directed against a specific antigen.
This is called antigen specific antibody. It is not commonly
available, and has greater specificity than is necessary for
‘most procedures.
'A fourth type of preparation, and one which is readily
available, is conjugated antibody (Figure 7). Conjugation is the
process of chemically inking some type of marker onto an an:
tibody molecule. This can be a fluorescent label such as,
fluorescein and rhadamine, or an enzyme such as alkaline
phosphatase or horseradish peroxidase. A wide variety of
Conjugates are avalable for use in various direct and indirect,
immunohistological stains.
Unfortunately, In the chemical process of conjugation,
‘small amounts of antibody and label can be destroyed. This,
ccan decrease the sensitivity and specificity of these reagents
{An alternative to artificially combining a marker to an antibody
Js an immune complex—the combination of an antigen and
its specific antibody utlizing the natural affinity they have for
‘one another. These complexes are specially prepared to re
‘main soluble and not to form precipitates in solution. An ex:
‘ample of ths isthe peroxidase antiperoxidase (PAP) complex.
Which consists ofthe enzyme peroxidase (the antigen) and an
antibody specific for peroxidase (Figure 8). The use of these
naturally formed immune complexes instead of chemical con-
jugates, makes PAP staining procedures as much as 1,000,
times more sensitive than immunofluorescence.
Monoclonal Antibodies
A single antigen molecule contains several characteristic
antigenic determinants or epitopes. When an antigen is
injected into an animal as described previously, the
Bulymphocytes will make antibodies against the antigen. One
B-cell can form antibadies against only one antigenic epitope.
‘Since there are many B-cells producing antibodies against
‘each epitope, this is called a polyclonal (many cells) antibody.
In some techniques it is desirable to have an antioody
‘specific fora single epitope. Since this is produced by a single
B-cell line (called a clone) it is termed a monacional (single
coll) antibody.
‘The selection ofthe B-cell clone producing the desired an-
tibody must be performed outside the animal. However, these
cells cannot grow and divide once removed from their host,
and wil die in approximately one week. In order fo preserve
the antibody production capebillies of the clone, the B-cells
fused with a myeloma cell (@ cancerous B-cell) that can ive
‘almost indefinitely outside the host animal. The hybrid
‘myeloma cell (hybridoma) that is formed by this fusion can be
{grown in cell culture as can the myeloma cell, and will pro-
duce the antioody that was being made by the B-cell
‘To cbiain a consistent supply of monoclonal antibody, the
hybridoma formed must be stable, First, the B-cell and the
‘myeloma cell must come from the same animal species. The
‘myeloma cell must be suitable for hybridizing and be easily
propagated in culture. & large number of Biymphocytes must
be avallable for fusion, The animal that most reacily fulfils
these criteria Is the mouse.
The frst step in producing a monoclonal antibody is iden-
tical to that of making a polyclonal antibody (Figure 9). A
‘mouse is injected with a purified antigen, and will begin mak-
ing anticody against i, When large amounts of antibody are
being produced, the mouse is sacrificed and the spleen, cor
taining large quantities of B-lymphocytes, Is removed. A cell
‘suspension is made and is mixed with the myeloma cells in a
‘medium that will cause the cells to fuse. Unfused and im
property fused cells wil die, while the desired hybridomas will
live and grow in culture, The hybridomas are tested to deter:
‘mine which clone is producing antibody against the desired
epitope. This is the most dificult and time-consuming part of
the procedure.
Once the appropriate cell line is identified, it can be in
Jected back into a mouse where it wil produce a tumor. The
ascitic fluid from the tumor will contain high concentrations of
the antibody, as well as other mouse immunoglobulins and
proteins which can cause increased background staining in
Immunoperoxidase techniques,
‘Another method of producing monoclonal antibodies Is 10
{grow the hybridoma in tissue culture, The supematant fluid
will contain the antibody produced by the hybridoma. Culture
supernatants contain lower concentrations of antibody than
ascitic fluid, but nonspecific background staining due to
‘undesired proteins is eliminated, Handbook of
ae
Figure 8. Procucing monocional antibodies.
rt
Asoc Fluid + Other proteins present
Tiseue Culture
‘Supernatant Fluid
> ° ‘ee
©0®
5 Oo 1
tmlora cor (@)
Antibody to Wl epitope
Antvody to epitope
Antibody to @ epitope
\ (e)
- 6
© Peroxidase Enzyme
> Biotin
Figure 10. Direct Method Figure 11, Increct Method,
Figure 12. PAP Method.
Figure 12. Aviin Biotin Method. lmmunoperoxidase Staining Methods
STAINING METHODS
‘There are four main methods of immunoperoxidase stain
ing that can be used to localize cellular antigens, The direct,
indirect, PAP, and avidin-biotin methods each have certain ad-
vantages and disadvantages which must be evaluated prior to
selection of the mast effective procedure for the work 10 be
performed,
Immunoperoxidase procedures allow visualization of cell
‘componenis in a variety of specimens including parafin sec-
tions, cryostat sections, smears, imorins and cytospins. For a
specific antigen it can be determined what type of cells pro-
‘duce this substance in normal and neoplastic tissue, levels,
of the substance produced, the identification of cells of
Unknown origin, and the ‘determination of tumor cell
dilerentiation,
Peroxidase
Immunoperoxidase staining involves the use of antibodies
‘and the enzyme peroxidase. Peroxidase is commonly used
for several reasons:
— Its small size will not hinder the binding of antibodies to
adjacent sites.
Is easily obtainable in highly puritied form so that the
chance of contamination is minimized,
— Itls very stable, and therefore will emain unchanged
during manufacture, storage and application.
— Only small amounts are present in issue specimens,
and this endogenous peroxidase activity is easily
quenched,
— There is @ wide availabilty of chromagens which can
be acted upon by peroxidase to form a colored end.
product that will precipitate atthe site ofthe antigen to
be localized.
— Its inexpensive.
Direct Method
‘The simplest way to localize a certain antigen is by using
‘an antibady directed specifically agains it. In the direct im-
munoperoxidase method this specific antibody is chemically
linked to peroxidase. The conjugated reagent is applied to the
specimen and will react with the antigen (Figure 10). A
substrate is then applied which will produce a colored end
product precipitating at the site, and thus mark the localized
antigen.
Direct technique can be performed very quickly, with low
probability of nonspecific reactions. The main drawback is
that for every antigen to be localized, a different conjugated
antibody is needed. Ifthe antibody cannot be obtained in con
jugated form, then the user must either perform the conjuga-
tion himself, or chose another procedure.
‘The most common application ofthe direct immunoperox-
dase method is for the detection of immunoglobulin, comple:
‘ment and immune complex deposits in kidney biopsies from
patients with various types of renal disease. These same an-
tigens can also be localized In skin biopsies from cases of
systemic lupus erythematosus (SLE) and other connective
tissue disorders. The most common antigens identified in
these cases are IgG, IgA, lgM, C3 and C4,
Indirect Method
Inthe indirect application, an unconjugated antibody will
bind to the antigen in the specimen, To localize this attach-
‘ment, a peroxidase conjugated antibody is needed to bind to
the first antibody. For example, i the primary antibody was
made in a rabbit, then the conjugated secondary antibody
‘must be specific for rabbit antibody. A substrate is added 10
localize the reaction (Figure 11).
This method Is rmore versatile than the direct method
because a variely of primary antibodies made in the same
‘animal species can be used with one conjugated secondary
antibody. Therefore, this procedure can be used to advantage
with any primary antibody when a peroxidase conjugated sec:
‘ond antibody is avaliable. However, the procedure takes ap-
proximately twice as long to complete as the direct method,
‘and there is greater chance of nonspecific reactions
‘occurring.
The primary use for the indirect immunoperoxidase tech:
rique i to identity antibodies in the serum of patients with
variqus autoimmune, bacterial and parasitic ciseases. In this
procedure, the patient's serum is applied to the antigen con:
taining specimen in place ofthe primary antibody. The perox-
idase conjugated secondary antibody is specific for human
immunoglobulin, I the patient has an antibody that can react
wih the antigen in the specimen, the peroxidase conjugated
ant:human immunoglobulin will bind to the patient antibody,
land show postive staining for the antigen. If the patient's, 2
serum contains no antibody to that antigen, the secondary an-
tibody cannot bind, and no staining of the antigen will be seen.
Some of the more common patient antibodies identified are
‘against nuclear, thyroidal, mitochondrial, and smooth muscle
antigens: treponema pallidum; herpes simplex virus; and
cytomegalovirus.
PAP Method
This method utilizes three reagents: Primary and secon:
ary antibodies, and PAP Complex—comprised of the en-
zyme peroxidase and an antibody against peroxidase. The
primaty antioody is specific for the antigen. The secondary or
“link” antibody is capable of binding to both the primary and
to the PAP Complex, because both are produced in the same
animal species.
Functionally, the link antloody is added in excess so that
only one of its Fab sites will bind to the primary, leaving the
other Fab site free to bind to the antibody in the PAP Complex.
The peroxidase enzyme is visualized via @ substrate:
cchromagen reaction (Figure 12)
‘Absence of conjugated antibodies in this method means
‘greater sensitivity than that attributed to the direct and in:
direct techniques. This Is especially evident in formalin fixed,
paratfin embedded tissues where strong staining can be
‘observed even though much of the antigen has been de-
stroyed by fixation and processing. Due to this loss of antigen
during fixation, the direct and indirect techniques must be
performed on frozen sections to achieve consistent results
‘The greater flexibility of the PAP method in specimen process-
ing seems to compensate for the increased time required by
‘this method.
‘One of the mast important applications of the PAP method
Is in determining the origin of tumors by identifying specific
antigens the cells produce. This allows for more accurate
Classitication—especially of poorly differentiated and
‘metastatic tumors—than can be achieved on the basis of
‘morphology alone. The fact that the PAP method is applicable
to routinely fixed, paraffin embedded material circumvents
the need for frozen tissue and also permits retrospective
studies.
‘Some of the tumor markers commonly used are prostate
specific antigen (PSA) to identity tumors of prostatic origin;
immunoglobulins such as kappa and lambda light chains, 196,
IgA and IgM to distinguish B-cell ymphomas from unditferen-
tiated carcinomas; glial fibrilary acidic protein (GFA) wich
siains all tumors of glial origin, both primary and metastatic;
‘and human chorionic gonadotrophin (HCG) which can be lo-
caalized in normal trophoblastic cells and the trophoblastic
element of germ cell tumors of the ovary, testis and ex
tragonadal sites.
Handbook of
Avidin-biotin Method
This method is based on the abilty of the egg:white
alycoprotein avidin 10 nonimmunciogically bind four mole:
‘cules of the vitamin biotin As with the PAP method, three
reagents are used. The fist is a primary antibody specific for
the antigen to be localized. The secondary antioody, capable
‘of binding to the firs, is conjugated fo biotin, The third reagent
is a complex of peroxidase conjugated biotin and avidin, The
{ee sites of the avidin molecule allow binding to the biotin on
the secondary antibody. The peroxidase enzyme, and there-
fore the original antigen, are visualized with an appropriate
chromagen (Figure 13)
Even though conjugated antibodies are used in this method,
the strong affinity of avian for biotin gives this method greater
sensitivity than other conjugated antibody techniques such as
the direct and indirect methods. Like the PAP method, ex
cellent results can be achieved on fixed, paratfin embedded
specimens,
“The avidin-biotin technique represents one of the most re:
‘cent developments in immunoperoxidase staining. It lends
itself to the localization of numerous antigens in a variety of
‘specimens. Much of the early work with this methad has been
in the identitication of pituitary hormones in normal and
neoplastic tissue, Other uses include staining for cell surface
markers, lectin binding sites, viral proteins and intermediate
filaments.
A
AI Wenn,
Figure 14, Peroxidase actvity already present in the
tissue wil show staining I not ined
before antiody application.
Figure 18. Nonspecific binding of primary antibody 10
‘charged collagen and connective tissue, wth
‘subsequent immunoperoxidase staining, can
be avoided by pretreatment of the specimen
wth animal serum, Immunoperoxidase Staning Methods
3
Endogenous Peroxidase Activity
‘The substrate-chromagen reaction used to visualize per-
‘oxidase cannot distinguish between the enzyme immunologi
cally localizing the cellular antigen, and similar enzymatic
activity present in the specimen before staining. This en-
dogenous peroxidase activity is confined mostly to red and
white blood cals. If itis not removed before adding the mark
ing enzyme, positive staining will be observed thal is due not
ta the specific antigan alone, but also due 10 peroxidase ac-
tivity already present in the specimen (Figure 14)
There are several ways 10 iteversibly inhibit endogenous,
peroxidase, and one of these techniques should be per-
formed at the beginning of the staining procedure. The
‘ickest and easiest technique isto place 4 to 6 drops of 3%
hydrogen peroxide directly on the specimen, and incubate for
five minutes betore rinsing off
‘Specimens containing large numbers of blood cells such
‘as cylospins and blood smears will show a violent reaction
with ydrogen peroxide due to the large amount of perox
‘dase activity present. The bubbling and fizzing that occurs
‘can damage the cellular morphology, s0 3% hydrogen perox-
ide should NOT be used on these specimens. A similar reac-
tion will also occur when 3% hydrogen peroxide is placed on
cryostat sections embedded in O.C-T.
‘Consequently for cytospins, blood smears and frozen sec:
tions, a different procedure must be used. These specimens
should be placed in a bath of hydrogen peroxide and
‘methanol for twenty minutes. The bath of 200 mi absolute
‘methanol plus 50 mi of 3% hydrogen peroxide must be made
up fresh just before use, The formula amount can be scaled
up or down depending on the number of specimens to be
tested.
There are various other solutions that can be used in place
cf hydrogen peroxidemethanol. Slides can be placed in a
bath of 100 mi of concentrated ethanol containing 0.2 ml of
‘concentrated HC! for 15 minutes. A bath of absolute methanol
with 1% sodium ritroterricyanide and 0.2% acetic acid can
also be used. A slightly longer procedure is to incubate the
slides in 0.1% phenylhydrazine for one hour at 37°C. A two-
step procedure involves incubation with 0.01 % periodic acid
for ten minutes followed by a solution of 0.1 mg sodlum bore-
hydride/t mi of water for ten minutes 10 reduce any alde-
hydes produced by the periodic acid
‘Most tissues do not contain large amounts of blood cel or
endogenous peroxidase, and activity is usually confined to
blood vessels. In these specimens the incubation with
hydrogen peroxide can be omitted, although when inter-
preting the staining patterns this must be taken into con-
sideration, In specimens that do contain many blood
cclls—such as bone marrow, placenta and spleen—removal
tf endogenous peroxidase is essential to correct interprete-
tion. These specimens also serve as excellent controls when
‘evaluating the effectiveness of new lots of hydrogen peroxide.
Nonspecific Background Staining
Positive staining of @ specimen that is not a result of
antigen-antibody binding is termed nonspecific background
Saining. The most common cause is attachment of protein to
highly charged collagen and connective tissue elements of
the specimen. Antibodies are proteins. Ifthe frst protein solu
tion applied to the tissue is the primary antibody, it can be
nonspecifically adsorbed to these charged sites. The second:
ary antibody can stil bind to the primary and the peroxidase
‘colar reaction will occur, Positive staining of these sites is due
‘ot to localization of the tissue antigen, but to nonspecific an-
tibody attachment to collagen and connective tissue (Figure
15)
“The most effective way to prevent this nonspecific staining
is to add an innocuous protein solution to the specimen
before applying the primary antibody. This protein wil fil the
charged sites, leaving no room for adsorption of the primary
antibody. Tne most common source of the protein solution is.
nonimmune serum from the same animal species that pro-
duced the secondary antibody. This avoids positive staining
due to binding of the secondary antibody to components in
the protein solution.
‘The nonimmune serum is used fairly concentrated at du:
tions of 1:5 to 1:20, Addition of 25% bovine serum albumin
(BSA) will increase the protein concentration and further
reduce the nonspecific staining. The nonimmune serum is ap-
plied to the specimen and allowed to remain for 10-20
minutes. The side is not rinsed, but excess serum is tapped
off leaving a thin layer coating the tissue when the primary an
tibody is applied. Care must be taken that only a small amount
of the often viscous serum remains or the primary antibody
will be diluted, resulting in pale staining of the antigen.
‘As with endogenous peroxidase activity, not all specimens
‘exhibit appreciable nonspecific binding, The incubation with
onimmune serum can be eliminated in these specimens as,
Jong as it does not interfere with interpretation. Other causes
‘of nonspecific backgraund staining such as inappropriate an:
tibody dilutions, incomplete removal of paraffin, improper
Finsing of slides and incorrect substrate incubation will be
plete penetration of fixative, resulting in nonspectic.
staining
— After fixation, tissue specimens should be thoroughly
washed to remove excess fixative which can cause
staining artifacts.
‘The most practical, and probably the best alharound fix-
ative Is 10% buffered formalin. Due to its crosstinking
characteristic, itis an especially good fixative for small an-
tigens—such as hormones. However, fixation time is critical
to prevent antigen masking. Immunoglobulins are especially
susceptibie to overfixation with formalin. In these cases, treat>
ment with proteolytic enzymes will digest the excess
aldehyde linkages and expose the antigen
Formalin should be fresh and buttered to a pH of 7.07.6.
{As this is a slow acting tative, acidic solutions can cause
structural disturbances and poor morphology. Due to the
problem of antigen masking, fixation should be for the
shortest possible time to achieve good morphology; usually
6-12 hours, Tissue fixed for longer periods of time may stil
exhibit intense staining depending upon the concentration of
antigen present.
10% Butfered Neutral Formalin, pH 7
Formalin (40% formaldehyde) 100m!
Dibasic sodium phosphate, anhydrous 65a
Monobasic sodium phosphate, monohydrate 4.09
Distilled water 900m
‘Another excallent fixative Is Zenker's fixative. It does not
reduce aldehyde linkages, and is often the fixative of choice
{or specimens to be stained for immunoglobulin, The fixation
time is short, usually 2-4 hours. To prevent black deposits on
the stained specimen, the mercuric chloride must be re-
‘moved afer the sections have been deparaftinized.
Zonker's Firative
Potassium dichromate 25mg
Mercurie chloride 50g
Sodium suliate 109
Distilled water 4,000m!
Glacial acetic acid 50m!
Dissolve the salts in the distiled water by heating and str
ring, Add the glacial acetic aci.
Removal of mercuric chloride crystals
1. Deparaffinize and rehydrate section to 95% alcohol
2, Place slide in 05% alcoholic iodine (0.59 iodine
Crystals in 100ml of 70% ethanol) for 3 minutes.
8. Gently rinse in running water.
4, Place slide in 6% sodium thiosulfate (69. sodium
thiosulfate in 100m distiled water) for 2 minutes
5. Gently rinse in running water
6. Continue with immunoperoxidase staining.
{A third fixative that is gaining in popularity is Bouin's. This
fixative produces good morphology and has been recom.
‘mended for localization of J-chains, immunoglobulins and
alphe-fetoprotein, The fixation time is the same as Zenker's;
Usually 2-4 hours. To prevent artifacts, excess fixative must be
removed tram the tissue by thorough washing in several
changes of 50% and 70% alcohol
Bouin's Fixative
19% saturated picrc acid 750m!
Formalin 40% formaldehyde) 250m
Glacial acetic acid om
For smears, cryostat sections, cytospins and imprints, tx
ation in acetone at 4°C for 10 minutes is recommended. As
‘an alternative, if endogenous peroxidase activity is quenched
in a hydrogen peroxidemetnanal solution, the methanol will
fix the specimen,
Processing
‘After fixation, tissue specimens can be dehydrated,
cleared and embedded according to routine processing pro- Immunoperoxidase Staining Methods
‘cedures. For best results, specimens should be embedded in
ure paraffin because it can be completely and easily re-
"moved from the tissue atthe time of staining. The plastic con-
tained in some embedding media is often dificult to remove,
and can cause nonspecific staining and inhibition of specific
siaining, Since most laboratories routinely use embedding
‘media containing plastic, certain precautions must be taken:
1. If the embedding media is heated to temperatures
above 62°C, the plastics will star to form polymers.
These polymers are very dificult to remove from the
tissue, and can also cause nicks in the microtome
knife. Consequently, paraffin baths should be kept at 2
maximum temperature of 60°C to prevent polymeriza-
tion,
2. To aid in complete removal of the embedding media,
slides should be placed in a 60°C oven for 30 minutes
prior to deparatfinizing, Temperatures must not exceed
60°C to prevent antigen denaturation and damage to
cellular morphology,
3. From the oven, the slides should be immediately
transferred toa fresh xylene or toluene bath, The parat-
fin should not be allowed to soliaty before deparattiniz-
ing as this will negate the benefits of melting. Either
xylene or toluene can be used to deparaffinize. Some
researchers feel that toluene is more effective for com-
plete removal of embedding media,
4, For efficient plastic removal, no mare than SO slides
should be run through a 250ml xylene bath before itis
changed. This may seem to be an insignificant point,
but itis critical to reducing the amount of background
staining. The fewer the number of slides processed
through a xylene bath, the more completely the embed-
ding media will be removed,
5. I more than 20 slides are placed in a 250ml bath at one
time, xylene circulation may not be sufficient. For this
reason, when processing several slides at one time, a
second, fresh xylene bath should be used.
Ital ofthese precautions are observed, there should be no
problem with nonspecific background staining due to plastic
residue. This type of interference is easly identified by pale
Staining that extends beyond the tissue specimen. All other
types of background staining are confined within the borders
of the spebimen.
Procedure for Depavifnizing and Rehycration
1. Gircle and label specimen with a ciamond pencil
2. Place in 60°C oven for 30 minutes,
8, Transter immediately to a fresh xylene bath for 3
minutes.
ar
4, Place in a second, fresh xylene bath for $ minutes if
desired,
55 Place in a bath of absolute alcohol for 3 minutes.
6. Place in a second, fresh absolute alcohol bath for 3
minutes.
7. Place in a bath of 95% alechal for 3 minutes.
8. Place In @ second, fresh 96% alcohol bath for 3
minutes.
8. Rinse under gently running water,
10. Begin immunoperoxidase staining
one and calcified tissue must have the insoluble calclum
salts removed before they can be sectioned. This causes no
problem in immunoperoxidase staining as long as. the
‘specimen has been thoroughly fixed. Adequate fixation will
prevent damage to the antigen and cellular structures by the
\decaleitying solutions. Commercially available decalcitication
reagents tend to give variable results In immunoperoxidase
staining, The most consistent results have been obtained us-
Ing @ 5% nitric acid solution for one hour. The specimens
should be cut into fairy small pieces to allow complete
Penetration of fixative and acid, After decaloifcation, the
‘Specimen should be washed in running water to remove ex-
‘cess acid before continuing with routine processing,
Adhesives
Due to the frequent handing and rinsing ofthe slides, the
tissue sections may become loosened and falloff. Various
methods can be used to prevent this. Several slide adhesive
solutions are commercially avalable, or they can be easily
made using the formulas given below. All of the adhesives
should be used sparingly as an excess can cause nonspecific
‘background staining similar to that of unremoved embedding
‘media.
(One of the best and easiest methods to adhere the tissue
to the slide is by thorough drying after sections are cut. Pac.
ing the slides in a 60°C oven for 30 minutes will melt the
paraffin and promote close contact between the tissue and
the glass slide. This important step should be performed even
If chemical adhesives are used.
Mayer's Egg Albumin
Separate an egg placing the white into a beaker. Discard
the yolk. Add an equal volume of glycerin and mix well. Filter
through coarse fiter paper. Add one crystal of thymol to pre-
vent bacterial growth, Store in the refrigerator. Handbook of
To use, place a small drop on the end of a slide and
istrioute evenly. Drying the slides in a 60°C oven for 30
minutes after the sections are cut will coaguiate the egg
albumin and adhere the specimen to the slide.
This adhesive should not be used if the specimen will be
trypsinized, as it will also be digested.
Elmer's Glue, 0.5%
Dissolve 0.5m! of Elmer's Glue-All Borden, Inc. Columbus,
‘Ohi) in 100m of distilled water heated to approximately
60°C. Place a small drop on the end of a glass slide and
distribute eveniy.
Chrome Alum Gelatin Adhesive
This is an excellent adhesive often used to assure
adherence of sections during enzyme digestion
1. Add 3:09 gelatin to one Iter of distilled water heated to
‘approximately 60°C.
2. Mix with magnetic stirrer until completely dissolved,
3, Add 0.5 grams chromium potassium sulfate (chrome
alum), Solution will appear blue.
4, Add several thymol crystals
5. Continue to stir until alum and thymol are completely
dissolved
6. Filter @ small portion while hot into a 100m! beaker.
Keep stock hot
7. Place several sides, one at a time, into the beaker.
Keep beaker 2% full at all times by replenishing with
hot stock solution.
8 Withdraw slides, ane at a time, and blot on edge of
towel, Place slides vertically na rack, and dry in a hot
oven.
9. When slides are completely dry, collect and place in a
slide box.
10, Slides should be stored at room temperature. They
can be used as needed
Instead of coating the slide with an adhesive, % tsp. of
gelatin andlor the same amount of Elmer's Give-All can be
‘added to the water bath used to float the tissue ribbons. The
water bath must be carefully cleaned after each use to pre-
vent bacterial growth,
‘Smears
‘Smears should be prepared carefully to assure that they
are only one cell layer thick. Thicker preparations wil in
terfere with interpretation as reagents become trapped be-
tween layers. Smears should be fixed as soon as possible
after preparation, or within 24 hours, The most common fx
atives are acetone or 10% buffered formalin for 10 minutes.
‘After fixation the specimens are usually stable for several
{days al room temperature, or one to lwo weeks when reriger
fated. When frozen at —20°0, the smears remain stable for
several manths. The ultimate preservation and stability of the
smear Is dependent upon the antigen to be localized.
Prior to staining, the specimen should be hydrated in bul
fer for § minutes before proceeding. Blood smears should be
placed directly in a hydrogen peroxide/methanol solution to
quench endogenous peroxidase activity. This will also serve
to fix the specimen, plus pre-hydration is unnecessary. The
slide Is incubated for 20 minutes in the solution, rinsed in
water, and stained in the usual manner.
Frozen Sections
Frozen sections are the specimens of choice for direct
land indirect staining methods. Most surtace antigens are
‘destroyed by fixation and can be localized only in frozen sec:
tions. To circumvent the detrimental effects of fixation and the
need far enzyme digestion, frozen specimens are olten used
{or the identification of intracellular antigens as well,
Specimens should be small in size and quickly frozen, This
‘allows for rapid fixation ofthe antigen and prevents the forma:
tion of ice crystals which disrupt cell structure. Once the
tissue is frozen, it can be stored at — 80°C for long periods of
time,
‘A piece of tissue no more than 4mm thick is placed in
aluminum Toll and immersed in @ dry icelethanol bath or an
isopentanelliquid nitrogen slush for 1-2 minutes or until
frozen. Prior to sectioning, the tissue biock is attached to a
metal specimen holder with a drop of water. Sections should
be cut 4:6 microns thick in a cryostat at ~ 20°C and fixed for
5 seconds in room temperature acetone, At this point the
tissue can be stored up to a month at 4°C, although for best
results staining should be performed shortly after sectioning,
Before beginning the immunoperoxidase staining pro-
‘cedure, the section is fied in acetone at 4°C for 10 minutes.
‘The slide is air dried and then placed in a buffer bath for §
minutes. If endogenous peroxidase activity is quenched using
a hydrogen peroxide methanol bath, the acetone tiation step
can be eliminated,
Plastic Embedded Sections
In addition to embedding tissue in paraffin, plastic com-
pounds may also be used. Plastic embedded specimens can
be cut in thinner sections than possible wih paratfin, and can lmmunoperoxidase Staining Methods
also be used in electron microscopy techniques. In this way,
antigen localization atthe light microscope level can be com:
pared with that of the electron microscope. The thin sections
also have the advantage of superior morphologic detail over
their paraffin counterparts.
Two main types of embedding media are available,
Methacrylates do not copolymerize with tissue components,
thereby preventing alteration of antigenic structure. These
plastics are often used when the antigen concentration is low.
Methacrylates tend to polymerize unevenly which can cause
shrinkage and subsequently poor morphology. The epoxy
resins such as Epon and Araldite polyermize more evenly
resulting in excellent preservation of structural detall
However, due to their high reactivity, they can interact with
tissue structures during the polymerization process.
Immunoperoxidase staining can be performed either
before the tissue is embedded (pre-embedding technique) or
afterwards (post-embedding technique). Each method has its
‘advantages and disadvantages, In the pre-embedding tech:
rique, the tissue is fixed and thick sections (5-50 micron) are
cut and either placed on slides or left free floating. The
specimen is stained in the usual manner except that longer in-
cubation times (2-24 hours) must be used to allow the an-
tibody to penetrate the thick specimen. To be certain that all
unreacted antibody is removed, the butfer baths are in-
creased to 20 minutes, with three separate baths between
each antibody incubation. After staining, the specimen is
postfixed for one hour in 1% osmium tetroxide, washed,
dehydrated and embedded in resin, Thin sections (1 micron)
are cut for use in light microscopy or ultrathin sections for
use in electron microscopy.
Long antibody incubations and buffer baths make this pro-
‘cedure time consuming to perform. Incomplete or uneven
penetration of antibody will result in poor staining, Special
‘care must be taken to thoroughly fix the specimen to prevent
the polymerization process from altering the morphology of
the stained ces. The main application for the pre-embeading
staining technique isin electron microscopy.
In the post-embedaing staining technique, specimens are
fixed, dehydrated and embedded in plastic before being
stained, For light microscopic examination, 1 micron thick
sections are cut and placed on slides. For electron
‘microscopy, ultra-thin sections are cut and picked up on
nickel or gold grids. To expose the antigenic determinants, the
Plastic must be etched before application ot the primary
antibody. The most common method is by incubation in a
solution of sodium hydroxide in absolute ethanol (sodium
‘ethoxide) or methanol (sodium methoxide) for 15 minutes. A
‘saturated solution of sodium hydroxide in absolute alcoho! is
prepared and allowed to mature for several days. Dilute 1:1 (1
part ofa tofal of 2 parts) with absolute alcohol before use. An
alternative Is to incubate the section in 10% hydrogen perox-
ide for 15 minutes. The slides are rinsed in water and the im-
Munoperoxidase procedure Is then continued in the usual
manner.
The post-embedding technique can be performed quickly
and easly with litle alteration in routine immunoperoxidase
‘methods. As with antigen localization in paratfin sections, the
‘specimen must be adequately ixed to survive plastic embed-
ding and polymerization. I the staining isto be viewed with an
electron microscope, a fixative shauld be chosen that wil
preserve ultrastructural morphology. The most commonly
Used fixative is 2% glutaraldehyde in 0.1M phosphate butler
pH 74. As this is a cross-inking fixative, it may be necessary
to perform an enzyme digestion after the plastic Is etched.
Immunoperoxidase staining of plastic, emibedded material
for either light or electron microscopy is a relatively new
technique. New modifications and uses are constantly being
