IMMUNOHISTOCHEMISTR

Y
SEMINAR 2
PREPARED BY: Camille Ann R. Castillo, RMT

HISTOCHEMISTRY
- a science that combines the techniques of
biochemistry and histology in the study of the
chemical constitution of tissues and cells.
IMMUNOLOGY
- a science that deals with the immune system,
cell-mediated and humoral aspects of immunity and
immune responses.
IMMUNOHISTOCHEMISTRY
- is the localization of a known antigen in
tissues by utilizing antibodies directed towards that

IMMUNOHISTOCHEMISTRY
(IHC)
Make

use of antigen- antibody

interactions

Site

of antigen binding is demonstrated

by direct labelling of the antibody, or by
means of secondary labelling method.

APPLICATIONS OF IHC
Tumor Pathology

Non- Tumor Pathology

Classification of
Neoplasma

Neurodegenerative

Diagnosis of
Malignancy

Brain

Prognostic Markers

Predicting response to
treatment

Detection of
metastases

diseases

trauma

Muscle diseases

Amyloidosis

Dementias

STEPS IN
IHC

I. TISSUE SECTIONS
(FIXATION)
Tissue must be prepared as a cryostat section
and fixed for a few seconds in absolute methanol
or acetone.
Why fix?

Helps to prevent: ELUTION, DEGRADATION &

MODIFICATION
Preserves
Provides

the position of the Antigen

target for Antibody molecules

FORMALDEHYDE

is the preferred fixative

I. TISSUE SECTION (SLIDE

PREPARATION)
2-4

micron tissue sections are cut into slides

Charged

sections

slides provide adhesion to tissue

The

tissues are further adhered to the slides by

baking at 60 degrees Celsius

DEPARAFFINIZATION
Tissue is treated in a series of xylene and
alcohol to remove paraffin

II. ANTIGEN RETRIEVAL

Enables

the partial reversal of formaldehyde induced

confirmational change of Ags.

Increases

the accessibility of the Ab to the Ag.

Choice

of Ag retrieval depends on the Ag to be

demonstrated.

METHODS:
Proteolytic

Enzyme Digestion

Microwave

Antigen Retrieval

Pressure

Cooking Antigen Retrieval

A. PROTEOLYTIC ENZYME
DIGESTION
Formalin

fixed paraffin sections are usually pretreated with proteolytic enzymes to break down
formalin cross-linking, unmask and allow certain
antigenic sites to be exposed.

Especially

useful to demonstrate HEAVY CHAIN

IMMUNOGLOBULIN, COMPLEMENT and SPECIFIC
ANTIGEN.

Most

common enzyme used are TRYPSIN AND

PROTEASE.

B.MICROWAVE ANTIGEN
RETRIEVAL
A

new technique that involves the boiling of formalin

fixed deparaffinised sections in certain solutions (0.01 Mcitrate buffer [ph 6.0], EDTA at ph 8.0 or Tris EDTA @ ph
9.9 or 10.0

Many

antigens can be retrieved.

Most

antigen retrieval methods apply temperatures near

the boiling point of water.

Care

should be taken not to allow the sections to dry

after heating, as this destroys antigenicity.

Fibrous

and fatty tissues tend to detach from the slide

(use strong adhesive like Vectabond)

C. PRESSURE COOKING ANTIGEN

RETRIEVAL
Another

alternative that appears to be less time

consuming

Allows

more consistent recovery of many

antigens (compared with microwave)

USE OF HIER (Heat Induced Epitope Retrieval)

Tissues

sections are heated to app 1000C

Achieved by:
Microwave oven
Pressure cooker
Vegetable steamers
Water bath
Automated Immunostainers
The cooling of sections slowly allows the protein to refold
properly
Protease Induced Epitope retrieval (PIER)

III. BLOCKING
Peroxide Block
Blocks endogenous peroxidases
3% H2O2
Protein Block
Blocks all non specific sites
Reduces background
10% Normal serum is used

IV. ANTIBODY (PRIMARY &

SECONDARY)
2 TYPES OF ANTIBODIES:
POLYCLONAL ANTIBODY
Produced by different cells of the animal; they are
immunochemically not identical to each other.
They react with various epitopes of an antigen;
may cross- react with other molecules and cause non
specific staining.
MONOCLONAL ANTIBODY
Products of an individual clone of plasma cells.
Hybridoma and cloning techniques have been

A. DIRECT METHOD
Conjugate

the primary antibody directly to

the label (FLUOROCHROME/ HORSERADISH
PEROXIDASE)

It

is simple and quick

It

is less sensitive
Which carries the risk of not detecting small
amounts of antigen that could be crucial in
making the diagnosis

Example: FITC, EPOS (Ehanced Polymer One-

DIRECT METHOD

B. INDIRECT METHOD
Involves

application of the unconjugated primary

antibody followed by labelled antibody directed
to the first antibody.

Relatively

inexpensive and more sensitive

Most

common enzyme used is HORSERADISH

PEROXIDASE

Can be:
Two-step Indirect technique
Three step Indirect technique

INDIRECT TECHNIQUE

B1. PEROXIDASE ANTIPEROXIDASE

(PAP) TECHNIQUE
An

indirect antibody enzyme-complex technique

Soluble

peroxidase- antiperoxidase complex is bound to

unconjugated primary antibody by a second layer of
bridging antibody that then binds to both the primary
antibody and the rabbit PAP complex.

Horseradish

for labelling

When

peroxidase is the most widely used enzyme

combine with the most common chromogen

diaminobenzidine (DAB) produce a dark brown
reaction end product when Ag is present.

B2. ALKALINE PHOSPHATASE ANTI ALKALINE

PHOSPHATASE COMPLEXES (APAAP)
Same

principle with PAP

Major

advantage compared to PAP is the lack of

interference from endogenous peroxidase activity.

Recommended

smears.

Endogenous

for use on blood and bone marrow

ALP is usually blocked by adding

levamisole to the substrate solution

B3. AVIDIN- BIOTIN COMPLEX

(ABC) TECHNIQUE
Uses

AVIDIN (derived from egg white) because of its

marked affinity for biotin (a low MW vitamin that can
be easily conjugated to antibodies and enzyme
markers)

Enzyme

that can be use is PEROXIDASE & ALKALINE

PHOSPHATASE

B4. LABELED STREPTAVIDIN AVIDIN

BIOTIN (LSAB) TECHNIQUE
Found

to be 4 to 8 times more sensitive than the old

ABC method.

Staining

sequence consist of:

Primary rabbit/mouse antibody

Biotinylated anti-rabbit/anti-mouse
Immunoglobulin
Streptavidin-Enzyme conjugate
Color

reaction is then developed with a use of a

chromogen/ substrate HORSERADISH PEROXIDASE

METHODS
Ag-Ab

label.

conjugates are visualized by the use of a

Enzymes that produce a colored precipitate in

the presence of a substrate are used as labels
Labels :
Peroxidase
Alkaline

Phosphatase

METHODS
Enzyme

labels produce a colored precipitate in the presence

of a specific substrate

Most

widely used label is Peroxidase

Produces

a dark brown precipitate when Diamino Benzidine

(DAB) is added.

Alkaline

phosphatase is also used and produces either red or

blue precipitates.

V. COUNTERSTAINING
Provides

contrast to the primary stain

Most

commonly used counter stain is Hematoxylin

and Eosin staining. It is considered to be gold
standard in IHC

Hematoxylin stains nucleic acids blue while Eosin

stains eosinophilic structures in shades of red, pink
and orange.

CONTROLS
Positive Controls:
Cells

or tissues that are known to contain the specific Ag

Detects
It

false negatives due to fixation and processing.

is used to validate the protocol or procedure used

Negative Controls:
Omission
Useful

of Primary Ab with the same tissue and procedure

to detect endogenous biotin and peroxidase activity

Internal Tissue Control:

Also

named as built-in control; eliminates tissue fixation

between specimens and controls

TROUBLESHOOTING (WEAK/ NO STAIN)

TROUBLESHOOTING (WEAK/ NO STAIN)

TROUBLESHOOTING (OVERSTAIN)

TROUBLESHOOTING (HIGH BACKGROUND)

END OF LECTURE