CIHRT Exhibit P-3116 Page 1

IMMUNOCYTOCHEMISTRY LECTURE'
(Also Called Immunochemis{ry ~r Immunoperoxidasel

I. Introduction
2. Antigens and Antibodies
3. Staining Methpds
4. Staining Procedures
5. Interpretation of Antibodies
 CIHRT Exhibit P-3116 Page 2
· .. ,

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: utiliziflg

antigen-antibody complexes. allow visualization of previously

undetectable cell components. This will guide you througlTJ

scientific knowledge necessary to perform immunoperoxidase

staining techniques.

We wi 11 start at the beginning by explaining exactly what

immunochemicals are. and wha t they can do. Once we' have an

understanding of the various methods that are used. we car' then

outline step-by-step procedures; discuss controls', {ixatio'] and

processing of specimens; and provide special. hints to achieve

successful sta£ning.
 CIHRT Exhibit P-3116 Page 3

ANTIGENS AND ANTIBODIES

It is necessary to have a basic knowledge of the building

blocks of immunology (antigens and antibodies) to more fully
understand immunoperoxidase methods.

Antigens
Antigens have two main properties. The first is
immunogenicity, which is the ability to induce antibody
formation. The second property is specific reactivity, which
means that the antigen can react "lith the antibody 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
immunohistochemical reactions are more prec'ise than ordinary·
histochemical techniques.
An antigen then. is a substance foreign to the host whic·h
stimulates formation of a specific antibody and which wi!l react
with the antibody produced. This reaction involves the formation
of immune complexes comprised of several antigen and antibo·dy
molecules. These complexes may be com eve r y I a I' g e a n d for m
precipitates which can be measured by various,techniques.

Antibodies
An antibody is a serum protein that is formed in response to
exposure to an antigen. and reacts specifically with that antigen
to 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 of this invader~
Antibodies are contained in the gamma globulin fraction of
serum, and are often called immunoglobulins (Ig). They can be
divided into five classes based on their size. weight, strgcrure,
function, and other criteria. The classes are IgA
(immunoglobulin A), IgO, IgE, IgG, and IgM. Antibody solutions
utilized in immunohistochemical staining. contain mostly IgG type
antibodies, with lesser amounts of the other classes.
 CIHRT Exhibit P-3116 Page 4

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, so the IgG molecule in Figure 1. will have
heavy chains of the gamma type. An IgA antibody has alpha heavy
chains; IgD, delta heavy chains. IgE, epsilon heavy chains; and!
IgM has mu heavy chains. A primary antibody for
immunoperoxidase staining that is "specific for gamma chains"
will localize the heavy chain of an IgG molecule.
There are only two types of light chains common to all five
groups: kappa and lambda. An IgG molecule has two identical
light chains. either two kappa chains or two lambda chains
(Figure 2). A single antibody can never have both kappa and
lambda chains. This is important when discussing the
interpretation of light chain staining in lymphoma cases.
The qr ienta t ion 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 antigen binding. or
Fab regions. These are the parts of the antibody molecule
capable of specifically binding to the antigen. This IgG
molecule has the ability to bind two antigen molecules, one at
each Fab site.
Antibody Production
In order to produce an antibody for laboratory use, it is
first necessary to purify an antigen. A source for the antigen
such as serum, urine or tissue is subjected to a combination of
procedures including precipitatio.n, 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
identify the antigen as foreign matter, and produce an antibody
directed specifically against it. Antibody production begins
within twenty minutes after injection, although a measurable
quantity of antibody cannot be detected for 5-10 days. Small
 CIHRT Exhibit P-3116 Page 5

blood samples are u,sually obtained and pooled 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
antigen used. housing 'facilities available .. amount of antibody
needed. and personal preference. Usually several animals of tln,e
species chosen will be injected with an anti.gen. as each anim.a,l
wil) vary in how it responds to the antigen. and in the am,o,u,nt
of antibody it produces. After several bleedings are p,o.lIlled:,'
contaminants present must be removed. This is usually
accompl ished by either liqu;id or sol id phase antigen ab:S0,rptlo.1'l
techniques.
 CIHRT Exhibit P-3116 Page 6

Heavy Chain Heavy Chain '

(gamma) (gamma)

Figure 1. IgG molecule showing paired heavy chains of gamma Figure 2. IgG molecules showing only possible light chain
type. configurations.

Fe

-~~A ~~ab-
AntigenA. Antigen

Figlue 3." Divisiori

Il-

or JgG molecule into Fc and FAb fragments.

--J
.
Figure 4. Whole serum antibody solution containing al/ normal
Antig~n binding occurs at the two FAbsiles. animal serum components in addition to specific antibody.

." figule a.-,mrpflnogJobuJin fraction of serum containing only Figule 6~ Antigen specific anUbOdy is more specific than tJS1:fijfJy
. : ;;rJi!XxJies. necessary.

Figule l A conjugate combines an antibody and some type of Figure B. A peroxidase-antiperoxfdase immune complex f-armed
visual marker. by natural affinities between antigens and antibodies;

Key 10 Figures 4 through B.

)~peeilie Antibody :: Other Antibody

..........::;:',
'
• Visual Marker •• Serum Components
........
...... '7 • Peroxidase Enzyme
 CIHRT Exhibit P-3116 Page 7

TYPES OF ANTIBODY SOLUTIONS

There are several antibody preparations available for use in

immunoperoxidase' procedures. The easiest to produce, and
therefore the most common and least expensiv.e, is whole serum.
Animal blood containing the antibody is centrifuged to separate
\he cells from the serum, and any contaminating antibodies are
absorbed out.
A whole serum solution is pictured schematically in Figure
4. It will contain antibodies specific for the antigen the
animal was immunized with. Other antibodies which are products
of the animal's normally functioning immune system will also 'be
present. These should not interfere wi th staining procedures.
The bulk of the whole serum fraction is made up of ordinary serum
components such a,s enzymes, electrolytes, and serum proteins,
Occasionally, these other serum elements can cause unwanted
background staining in some techniques. 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, cal lin:! an immunoglobulin
or Ig fraction, is depicted in Figure 5. This solution contains
mostly antibodies, both specific and naturally occurring, plus a
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 possible, as shown in Figure 6, to prepare a solution
containing only 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 linking some type of marker onto an
antibody molecule. This can be a fluorestent label such as
 CIHRT Exhibit P-3116 Page 8

fluorescein and rhodamine. or an enzyme such as alkaline

ph 0 s ph at a se 0 l' h 0 l' S era dis h per 0 x ida s e . A wid e val' i e t y 0 f
conjugates are available f~r use In various direct and indirect
immunohistological stains.
Unfortunately, in the chemical process o~ conjugation. small
amounts .
of antibody and label can be destroyed.
'
This can,
decrease the sensitivity and specificity of these reagents, An
alternate to artificially combining 'a marker to an antibody is an
immune complex - the combination of an antigen and its specific
antibody utilizing the natural affinity they have for one
another. These complexes are specially prepared to remain
soluble and not to form precipitates in solution. An example of
this is the peroxidase antiperoxidase (PAP) complex which
consists of the enzyme peroxidase (the antigen) and an antibody
specific for peroxidase (Figure 8). The use of these naturally
formed immune complexes instead of chemical conjugates, makes PAP
staini'ngprocedures as much as 1,000 times more sensitive than
im~unofluorescence.
 CIHRT Exhibit P-3116 Page 9

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 ,B-Iymphocytes wi 11
ma k e ant i bod ie's a g a ins t the ant i g en. 0 neB - eel I can for m
antibodies against only one antigenic epitope. Sillce there are
many B-cells producing antibodies against each epitojiJ.e. this is
called a polyclonal (many cells) antibody.
In some techniques it is desirable to have all antibody
specific for a single epi tope. Since this is prodl!lPceCI! by a
single B-cell line (called a clone) it is termed a monoclonal
(single cell) antibody.
rhe selection of the B-cell clone producing the desired
antibody must be performed outside the animal. However. these
cells canno't grow and divide once removed from their host, and
will die in approximately one week. In order to preserve the
antibody production capabilities of the clone, the B-cell is
fused with a myeloma cell (a cancerous B-cell) that can live
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 wnl produce the
antibody that was being made by thl B-cell.
To o'btain 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. A large number of B-lymphocytes must be available for
fusion. The animal that most readily fulfills these criteria is
the mouse.
The first step in producing a monoclonal antibody is
identica.l to that of making a polyclonal antibody (Figure 91, A
mouse is injected with a purified antigen. and will begin making'
antibody against it. When large amounts of antibody are being
produced, the mouse is sacrificed and the spleen. containing
large quantities of B-lymphocytes. is removed. A cell suspension
 CIHRT Exhibit P-3116 Page 10

is made and is mixed with the myeloma cells in a medium that will
cause the cells to fuse. unfused and improperly fused cells will
die, while the desired hybridomas will live and grow in culture.
The hybridomas are tested to determine which clone is producing
antibody against the desired epitope. This is the most difficult
and time-consuming part of the procedure.
Once the appropriate cell line is identified, it call be
injected back into a mouse where it will produce a tUlllor. 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 to grow
the hybridoma in tissue culture. the supernatant fluid will
•
contain the antibody pr'oduced by the hybridoma. Culture
..
supernatants contain lower concentrations
'
of antibody than
asciticfluid,'but nonspecific background staining due to
undes.ired proteins is eliminated.
 CIHRT Exhibit P-3116 Page 11

.....
·B· '" '-
Epitopes

..
.......-
... AntIgen ~. y-- ~ ~Mouse Spieen

• ~..• .......1C:....-.-..;:r-~~
,
';

FII/ure 9.l'tooucing monocional antibodies.

A!ilmbody !o • epito:;Je.
Aiil$ibod¥ .t;o . . efiJitr;r:Je:
AlIIibody t;o • ~

• 1\Mouse Tumor
~
~+411 ,
+.+ -¢
+.+.+
A+A+A
~+- .Other proteins present
Ascitic Fluid
at
@JJ
@
Tissue Culture Selection of Desired Clone
SUpematant Fluid

Key

AA Tissue Antigen • Petoxidase Enzyme

A
r
PtimatY Antibody

SecondatY Antibody
~ Biotin

A
y
X

PAP COmplex
Avidin

,. , ~
-.
~
,
~ ...•

A
A /\A
1\1\..
A
I\.A.
Figure 10. Ditect Method. Figure 11. Inclirect Method. Figure 12. PAP Method. Figure 13. Avidin·Biolin Method.
 CIHRT Exhibit P-3116 Page 12 I':}
7
STAINING METHODS

There are four main methods of immunoperoxidase. staining

that can be used to localize cellular antigens. The direct,
indirect. 'PAP, and avidin-biotin methods each have certain
advantages and disadvantages which ~ust be evaluated prior to
selection of the most effective procedure for the work to b·e
performed.
Immunoperoxi~ase procedures allow visualization of cell
components in a variety of specimens including paraffin sectio8£.
cryostat sections. smears, imprints and cytospins. For a
specific antigen it can be determined what type of cells produce
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 differentiation.
 CIHRT Exhibit P-3116 Page 13

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.
I t is easi ly obtainable in highly puri fied form so. that. the
chance of contamination is minimized.
It is very stable. and therefore will relliain lllilchang,e'd
during manufacture. storage and application.
Only small amounts are present in tissue specimens. and this
endogenous peroxidas~ activity is easily quenched.
There is a wide availabili.ty of chromagens which can be
acted upon by peroxidas~ to form a colored end product that
will precipitate at the site of th~ antigen to be localized
It is inexpensive.
 CIHRT Exhibit P-3116 Page 14

DIRECT M.ETHOD

The simplest way to localize a certain antigen is by using

an antibody directed specifically against it. In the direct
immunoperoxidase method this specific antipody'is chemically
1 inked to peroxidase. The conj uga ted reagent is appl ied 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. If the antibody cannot be obtained in
conjugated form. then the user must ei~her perform the
conjugation himself, or choose anOther proced~~~.·;
The most common application ofthedirij'ct' immunoperoxidase
method is for the detection of immunoglobuLin; complement and
immune complex deposits in kidney biopsies' from patients with
various types of renal disease. Thes~ same antigens can also be
localized in skin biopsies from cases· of systemic lupus
erythematosus (SLEJ and other connective tissue ·disorders.The
most common antigens identified in these cases are IgG; IgA., IgM;
C3 and C4.
 CIHRT Exhibit P-3116 Page 15

INDIRECT METHOD

In the indirect application, an unconjugated antibody will

bin-d to the antigen in the specimen. To localize this
attachment, a peroxidase conjugated antibody is needed to bind to
the first antibody. For example, if the primary antibody was
made in a rabbi t, then the conjugated secondary antibody\' must be
specific for rabbit antibody. a substrate is added to localize
the reaction (Figure 11).
This method is more versatile than the direct method because
a variety 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 conj uga ted second ant i body is avai lable.
However, the procedure takes approximately 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 technique
is to identify antibodies in the serum of patients with various
autoimmune, bacterial and parasitic diseases. In this procedure,
the patient's serum is applied to the antigen containing specimen
in place of the primary antibody. The pe_roxidase conjugated
secondary antibody is specific for human immunoglobulin. If the
patient has an antibody fha.tean react with the antigen in the
specimen, ·the peroxidase conjugated anti-human immunoglobulin
will bind to the patient antibody, and show positive staining for
the antigen. If the patient's serum contains no antibody to that
antigen, the secondary antibody 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 pallidurn;
herpes simplex virus; and cytomegalovirus.
 CIHRT Exhibit P-3116 Page 16

PAP METHOD

This method utilizes three reagents: Primary and secondary

antibodies. and PAP Complex - comprised of the enzyme peroxidase
and an. antibody against peroxidase. The primary antibody is
specific for the antigen. The secondary or "link" antibody is
capable of binding to both the primary and to the PAP Complex,
be~ause both are produced in the same animal species.
Functionally. the link. antibody is added in excess so that
only one of its Fab sites Idll bind to the primary. leaving the
.
other Fab site free to bind to the antibody in the PAP Comolex.
.
The peroxidase enzyme is 'visualized via a substrate-chromagen
reaction (Figure 12).
Absence of conjugated antibodies in this method means
greater sensitivity than that attributed to the direct and
indirect techniques. This is especially evident in formalin
fixed. paraffin embedded tissue where strong staining can be
observed even though much of the antigen has been destroyed by
fixation and processing. Due to'this loss of antigen during
fixation, the direct ~nd indirect techniques must be performed on
frozen sections to achieve coni~stent results. The greater
f lexibi I i ty of the PAP method in specimen processing seems to
compensate for the increased time r,equired by this method.
One of the most 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
classification 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 (PSAl to identify tumors of prostatic origin;
immunoglobulins such as kappa and lambda 1 ight chains. IgG. IgA
and IgM to distinguish a-cell lymphomas' from undifferentiated
carcinomas; glial fibrillary acidic protein (GFA) which stains
 CIHRT Exhibit P-3116 Page 17

all tumors of glial origin, both primary and metastatic; and

human chorionic gonadotrophin (HCG) which can be localized in
normal trophoblastic cells and the trophoblastic element of germ
cell tumors of theo~arY. ~estis and ex~ragonadal sites.
 CIHRT Exhibit P-3116 Page 18

ENDOGENOUS PEROXIDASE ACTIVITY

The substrate-chromagen reaction used to visualize

peroxidase cannot distinguish between the enzyme immunologically
localizing the cellular antigen. and similar. enzymatic activi ty
present in the specimen before staining. The endogenous
peroxidase activity is confined mostly to red and white blood
ce lIs. If it is not removed before adding the marking enzyme.
positive staining will be observed that is due not to the
specific antigen alone. but also due to peroxidase activity
already present in the specimen.
There are several ways to irreversibly inhibit endogenous
peroxidase. and one of these techniques should be performed at
the beginning of the staining procedure.
Most common method of blocking endogenous peroxidase is the
use of 0.3% HZOZ in absolute methanol. This method insures that
little or none of the antigen sites are lost.
 CIHRT Exhibit P-3116 Page 19

ENZYME DIGESTIO~

Overfixation of tissue specimens in formalin causes

formation of an excess of aldehyde linkages which will mask the
tissue antigen and prevent its localization by the primary
antibody. This is a particular problem when staining lymph nodes
for immunoglobulin. To unmask the antigen sites, the aldehyde
bonds can be digested with proteolytic enzymes.
The most common enzyme used is trypsin. It is the least
destructive to the tissue, and its reaction can be easily
controlled. Trypsin, like many other enzymes, requires certain
temperature and pH values for optimal activity. The incubations
0
are carried out at 37 C using prewarmed buffer on prewarmed
slides. Temperatures below 37 0 C will reduce the activity of the
enzyme.
Masking Antigenic sites
fixation so well preserves the tissue integrity that the
large molecules used in these techniques are prevented from
penetrating the tissue to the antigenic sites.
fixation works by producing cross linkages with proteins
thereby "masking" the antigenic sites.
use of tryp.sin @ 37 0 C proteolytic (enzyme) breaks these
cross linkages and releases antigenic sites.
it is the least destruction to the tissue.
 CIHRT Exhibit P-3116 Page 20

NONSPECIFIC BACKGROUND STAINING

Positive staining of a specimen that is not a result of

antigen-antibody binding istetmed nonspecific background
staining. The most common cause is attachment of prote·in to
highly charged collagen and connective tissue elements of the
specimen. Antibodies are proteins. If the first p.ro.teil!
solution applied to the tissue is tne primary antibody, n can be'
nonspecifically absorbed to these charged sites. The SeCOJilidary
antibody cab still bind to the primary and the peroxidase color
reaction will occur. Positive staining of these sites is due not
to localization of the tissue antigen, but to nonspecific
antibody 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 will fill the
,
charged sites, leaving no room for absorption of the primary
•
antibody. The most common source of the protein solution is
non immune serum from the same animal species that produced the
secondary antibody. This avoids positive staining due to bi.ding
of the secondary antibody to components in the protein sol~tion.

Poor fixation - fix properly overn~ght.

Drying of section -' only deal wi th a few slides at a time
when adding antisera, when sections are allowed to dry. false
positive staining occurs.
Improper dewaxing of sections allow to heat in 56 0
incubator overnight and de,wax wi tt) 4 changes of xylene - 10
minutes each.
 CIHRT Exhibit P-3116 Page 21

P.A.P.
Day I

Prepare Slides:
- Cut sections @3 microns on clear glass slide.
- Place in 58-60 degree C. incubator overnight
Day II

- Place 200 ml 1/10 PBS in a 37 degree c. incubator.

- Weigh out 0.2 gm trypsin.
- Make up albuminized 1/10 PBS:
- 1 ml bovine albumin per 100 ml 1/10 PBS. (This
solution is used to prepare all antibody dilutions and
for washing slides).
Procedure
(1) Transfer slides directly from 58-60 degree. C. incubator to:
1. xyl ene ...............•... 10 min.
2. xylene .. 10 mil).
3. xylene . 10 J!Un.
4. xylene .. 10 min.
5. . 95% al cohol . 5 min.
6. Cold Tap H20 ............• 5-1 ID miLl!.•
7. 37 degree Tap H20 .......• 3-5 aim;.

(2) . Add 0 . 2 gm trypsin to 200 ml l/lIDiEB'S in 37 degree It:

inc·ubator.
(3)cTransfer slides to trypsin solution for 6 minutes at 37
'degrees C. (Discard after use).
(4) Rinse in cold tap H20 for 5 min.
Inhibition of Endogenous Peroxidase
(5) Transfer slides to freshly prepared 200 IIll of methanol + 2
ml 30% H202 for 30 minutes
(6) Rinse in cold tap water for 5 minutes
(7) 95% alcohol, 5 min.
 CIHRT Exhibit P-3116 Page 22

- 2 -

(8) Abs. alcohol .............. 5 niin.

(9) Chloroform ................ 5 min.

(10) Acetone . . . . . ... . . . .. . .. . . . 5 min .

(ll) Cold Tap H2O .............. 5 min.

(12) Albuminized PBS ............ 5 min. minimum

Primary Antibody
(13) Prepare dilutions of primary antibody using albuminized PBS
140 - 200 ul per slide.
(14) Prepare slides one by one by drying outside and leaving a
film of the albuminized PBS around the section (NO AIR
BUBBLES). (DO NOT LET THE SECTIONS DRyl).
(15) Cover section with diluted primary antibody (one slide at a
time) for 30 minutes in a humidification chamber.
(16) wash each slide carefully with 1/10 PBS (albuminized) using
a squeeze bottle.
(17) Transfer to a staining dish of the albuminized PBS.
Secondary Antibody
(18) Prepare dilution as follows:
for 20 slides
PBS 1/10 - 2700 ul
Normal Human - 300 ul
Serum
Shake well and discard 200 ul of this solution.
Add 200 ul peroxidase conjugated rabbit immunoglobulines to
mouse immunoglobulins. Mix·well.
(19) Prepare slides as for primary antibodies and add secondary
antibodies for 30 minutes.
(20) Wash with albuminized PBS and return to staining dish of
PBS.
 CIHRT Exhibit P-3116 Page 23

- 3 -

Teritary Antibody
(21) Prepare dilutions as for secondary antibody using peroxidase
conjugated swine immunoglobulins to rabbit immunoglobulins.
(22) Prepare slid~s as before and add antibody for 30 minutes.
(23) Wash well with PBS and return to staining dish 0.£ PBS.
Revelation of Peroxidase Staining:
(24) Prepare DAB:
Dissolve 6 mg of DAB (diarninobenzidine) for 6 slides in 1@
ml Tris-HCL (0.05M). Add 3 ul of 30\ H20i just before use.
Keep in dark. (If larger quantities are needed, use several
tubes ready for H202.

d
10 ml
d
10 ml
!J
10 ml
(25) Arrange slides in humidification chamber. Put DAB on each
slide for 3 to 5 minutes. (Do not attelllpt to reveal too
many slides at one time - maximum 15 slides).
(26) Rinse with old tap H20 to stop reaction.
(27) Return to ~ staining jar of water.
Cl!unterstain
(28) Haematoxylin for 10 to 20 seconds
(29) Rinse in cold tap H20.
(30) 95% al cohol
(31) Absolute alcohol
(32) Acetone
. (33) Xylene
Mount with Permount
 CIHRT Exhibit P-3116 Page 24

INTERPRETATION OF RESULTS

E:-lA
1 S '0 f val u e inc 0 n fir min 9 the e pit h eli a l o r i gin 0 f a ri
anaplastic tumor.
should be used in conjunction with LCA
distinction can be drawn between carcinomas I+EMA -LeA} aDd
lymphomas l-EMA +LCA)'.
plasma cells stain +VE
also of. value for detecting metastatic CA cEdIs in sections
of lymphoid tissue.

LCA
also of value-.in confitming origin of anaplastic tumors
plasma cel~s - VE
lymphoid cells +
+ staining wi th LCA indicates the tumor is of white cell
origin and ,likely to be a lymphoma.

UCH L - 1
the great majority of T-cell lymphomas is lab~lled by UCHL-
1.
• VE thymocyte!; <subpopulation of T-cells) and mature T-
cells.
useful for subclassing lymphomas.
normal B-cells ate negative.

L-26
B-cell lymphomas are labelled by L-26.
useful for subclassing lymphomas.
reacts with B~cells.
 CIHRT Exhibit P-3116 Page 25

Monoclonal & Polyclonal·antibodies •.

a) Paraffin section immunohistochemistry
MONOCLONAL (3-step immunoperoxidase)
Intermediate & Microfilaments
anti-cytokeratin - KLl (55 kD) -Immunotech
- MNF1l6 - private source (Chittali.)
anti-vimentin - Dako
anti-desmin - Dako
anti-neurofilament - Dako
anti-GFAP - Dako
HMB 45 - Dako (melanoma cells)

Other
anti-CEA - private source (Ford)
anti-EMA - Dako
Hormones
anti-thyroglobulin - Monosan
anti-calcitonin - Dako·
Kit - Dako
Insulin
Glucagon
Somatostatin

Lymphocytes'
. .. . '.
; ~

LCA (CD45) - ·leukocytecollllllon. :,....Dako

....
UCHLl (CD45RO) - T-cell ...."
MTl (CD43) - T-cell
monocytes, macroPhages,
B-Iymphoblast
L26 (=CD20) - B-cell
LNl (CDw75) - B-cell (GCC) . - Clonlab
- rbc, epithelium
DNA7 LNl-like - pr.source
DBB42 - B-cell differentiation
- pr.source
DBA44 - mantle zone B-cells
monocytoid B-cells, hairy cells
- pr.source
LN2 = MB3 (CD74) - HLA-DR (cyto)
DND53 (CD74 like) " - pro source
 CIHRT Exhibit P-3116 Page 26

MB2 - B-cell (cyto) - Clonlab

endo/epiwea)c
Lymphocyte activation
BerH2 (COJO) - RS cells, ALC, - Oako
embryonal ca
anti-EMA - see above
other
\
Leu H1 (C01S) - x-hapten
granulocytes, RS cells,
epithelium - Becton-Dickimsom
Leu-7 - NK cells,
myelin basic protein - •
Factor VIII - endothelial cells
BNH9 - BG-related
endothelial cells - private source
anti IgM - Oako
anti-kappa "
anti-lambda "
POLYCLONAL (PAP) all from Oako
anti-IgGh
anti-IgMh
anti-IgAh
anti-kappa
anti-Ialllbda
anti-fibrinogen
anti-lysozyme
anti-~lpha~l~anti trypsin
anti-alpha~1~fetoprotein
anti-B2 microglobulin
anti-myoglobin
anti-TSH
anti-ACTH
anti-prolactin
anti-HCG
anti-GH
S-100
anti-NSE
anti-chromogranin A
anti-GFAP
anti-cytokeratin
anti-prostate specific aniigen
anti-prostatic acid phosphatase