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IMMUNOHISTOCHEMISTRY
ANJAN KUMAR.K.R.
PhD Scholar
Dept. of Veterinary Pathology
Veterinary College, Bangalore
KVAFSU
What is immunohistochemistry
History:
Applications of immunohistochemistry
Histogenesis and differential diagnosis of
neoplasms
Likely site of origin in adenocarcinomas
Quality assurance in sentinel lymph node
biopsies
Definition of transformation pathways
Detection of microorganisms
Prognostic and predictive markers
Principle of Immunohistochemistry:
antigen
IMMUNOHSITOCHEMISTRY STEPS
Fixation
Tissue sections
Antigen retrieval
Blocking endogenous enzymes
Blocking background staining
Primary antibody
Secondary antibody
Chromogen Substrate
Counterstain
Mounting
Microscopy
Slide 3 Observation
of 23
Fixatives
For preservation of cells and tissues in as
reproducible and life like manner as possible.
They denature proteins by coagulation or by
forming additive compounds and may be
both.
.
Formaldehyde
Paraformaldehyde
Glutaraldehyde
NBF
FORMALIN SUBSTITUTES
Bouins solution: deleterious effect on
CD5,CD10&cyclin
B5 lymph node biopsies, recommended for
cytoplasmic antigen detection,CD5 IS
AFFECTED
Zenkers solution- poor penetration,
Hollandes fixative- not a good fixative for
immunohistochemistry
Zinc formalin-for nuclear morphology, tissues
do not adhere to slides
Coagulative fixatives
Ethanol
Acetone
Tissue adhesives
Deparaffinization
Xylene + alcohol
Xylene substitutes
One step deparaffinization and antigen retrieval
method
Antigen retrieval
Is a high temperature heating method to
recover the antigenicity of tissue sections that
had been masked by formalin fixation
AR is influenced by temperature and Ph.
Principle
Calcium induced modification of protein
conformation
Calcium formed a complex with proteins in
formalin fixed tissues leading to antigen
masking
So high temperature and calcium chelation is
required to release calcium form this cage like
calcium complex
Ultimately involves re naturation or at least
partial restoration of proteins to its native state
by hydrolysis of cross links driven by heat
AR METHODS
PIER- protease induced epitope retrieval
Other enzymes like: trypsin, proteinase,
pronase, pepsin
Acts by digestion of proteins but cleavage is
non specific and some antigens may be
negatively affected.
Has low no. of antigens for which this method is
optimal
AR
HIER involves placing slide in heated solutions of
various composition before application of antibody
Involves shorter heating times with high temperatures
Methods: microwave, vegetable steamer, pressure
cooker
TYPES OF AR SOLUTIONS
0.1M Citrate buffer solution pH: 6.0
O.1M EDTA buffer solution pH: 8.0
0.5M Tris base buffer pH: 10.0
Washing buffers
Most laboratories employ PBS / TBS.
Adding tween 20 prooves to be more effective.
However PBS is not recommended when using
Alkaline phosphatase based detection system.
ANTIBODIES
Most commonly used are IgG
IgM is less commonly used.
TYPES OF ANTIBODIES
Monoclonal
polyclonal
POLYCLONAL
MONOCLONAL
Antigen
An antigen is a foreign substance that stimulates antibody
formation and has the ability to bind to an antibody.
Handling of antibodies
Storage and containers used:
Should have negligent protein adsorptivity
Poly propylene or borosilicate glasses.
Storage temperature:
Ready to use antibodies are stored at 2-8 C
Cocentrated antibodies are aliquoted and
stored at -20C
1:50
1:400
1:100
1:800
1:200
SECONDARY ANTIBODIES
Monospecific antibodies recognize antibodies
raised in one species
Multi link secondary antibodies- universally
recognize both mouse and rabbit antibodies
and are based on polymer back bone.
Conjugated secondary antibodies.
(2)
Polymer based secondary antibody
(1)
antibody 1, mouse
METHODS OF
IMMUNOHISTOCHEMISTRY
Direct method
enzyme
primary Ab
antigen
Indirect method:
Two step indirect method
Three step indirect method
primary Ab (mouse)
antigen
enzyme
substrate-chromogen
primary Ab (mouse)
antigen
PAP method
APAAP method
ABC - Procedure
LSAB METHOD
Uses enzyme-conjugated streptavidin.
Streptavidin is conjugated to several molecules
of enzyme horseradish peroxidase (HRP) or
alkaline phosphatase (AP).
The secondary antibody is conjugated to
numerous biotin molecules, each of which can
potentially bind to an enzyme-conjugated
streptavidin.
Double immunohistochemistry
Spectral imaging
Spectral imaging
sections.
Control:
Negative control:
using only one type of epitope retrieval for all
slides, only one negative control slide
needs to be run for every different tissue
block that is stained.
It is particularly important that the
negative control slide be subjected to
the same epitope retrieval procedures as
the primary antibody, since these epitope
retrieval procedures can markedly intensify
endogenous biotin artifacts.
ARTIFACTS IN IHC
Desquamartifact
"Bubble" artifacts
Drying artifacts
Trapping artifacts
Edge artifacts
Artifacts of poor fixation.
Bacterial contamination artifacts
Endogenous biotin artifacts
Graphite pencil artifacts
CONCLUSION:
logical differential diagnosis based on the
clinical and morphologic findings.
garbage in, garbage out.
IHC is not perfect and all tumors will not
necessarily react as they are "supposed" to
NEVER use IHC to tell you whether
something is benign or malignant
Thank you