Documente Academic
Documente Profesional
Documente Cultură
Pathology
COURSE
COURSE TEACHER:
DR.M.L.SATYANARAYANA,PhD.,
PRESENTED BY :M.VETRIVEL
Immunoperoxidaseis
refer
to
sub-class
immunohistochemical
or
History
1941: Coons et al., have demonsrated antigens on
tissue sections by using an antibody which was linked
to an fuorescent label
1966: Nakane and Pierce reported the use of
secondary antibody which was conjugated with
peroxidase enzyme.
1979:
Sternberger
described
the
peroxidaseantiperoxidase method.
1981: Hsu et al., have defined the method of avidinbiotin-peroxidase complex which is abbreviated as ABC
method.
Principle
Antigen-antibody reaction taking place
For visualization/detection, an enzyme known as
a peroxidase is used to catalyze a chemical
reaction convert colorless chromogens into
visible colored end-products
Reaction can be detected by light microscope
Types of IPT
Direct IPT
Indirect IPT
PAP complex IPT
ABC IPT
Immunolabelling methods:
1. Direct method: The antigen directly binds to its
Direct method
A substrate-chromogen solution is added producing
a colored end-product
substratechromogen
Labeled
Antibody
Indirect method:
Two step indirect method
Three step indirect method
Immunolabelling methods:
Indirect method:
i) Two step indirect method
Primary antibody is unlabelled. A secondary antibody (which is labeled)
is used.
Secondary antibody must be raised against the immunoglobulin
of the species which the primary antibody is made in.
Example If the primary antibody (which is now the antigen) is made in
mouse, the secondary antibody must be against mouse immunoglobulin.
substratechromogen
Methods include
peroxidase-antiperoxidase
(PAP)
alkaline phosphatase-antialkaline
phosphatase (APAAP)
The secondary antibody must be directed
peroxidase-antiperoxidase (PAP)
chromogen
Mouse
Mouse monoclonal
Peroxidase
antiperoxidase
(PAP)
ABC - Procedure
Immunoperoxidase Steps
Tissue sections
Antigen retrieval
Blocking endogenous enzymes
Blocking background staining
Primary antibody
Secondary antibody
Chromogen Substrate
Counterstain
Mounting
Microscopy
Slide 3 Observation
of 23
Type of Fixation
Coagulant Fixatives
Types:
denatured protein
in alcohol
Non-Coagulant Fixatives of
Cross-Linking Type
H 2C = O +
H 2O
HOCH2OH
Special Fixatives
Formaldehyde-based fixatives
Neutral buffered formalin
Bouins solution (picric acid, glacial acetic
Mercuric chloride based fixatives
B5 (mercuric chloride, formalin, sodium ace
Zenckers Solution (mercuric chloride, glac
acetic acid, potassium dichromate, sodium
sulfate)
Acetic acid-zinc chloride (formalin, glacial acetic
Periodate-lysine-paraformaldehyde
Ethanol
Acetone
Protein +Formalin
AR: Heat
in Water
Chemical Modification
of Protein Confirmation
(Crosslinking)
Hydrolysis
Re-Modification
(Restoration of Ag)
Strong Intensity of IPT
AR-IPT
Ab
Ag
Ab
Ag
Influence of Temperature on
Antigen Retrieval
no heat
Influence of pH on Ag
retrieval
The retrieval solution pH is important.
pH4
pH7
pH9
no retrieval
proteinase Ktrypsin
Monoclonal Antibodie
Produced mostly in mice
Hybridoma technology = ascites vs. cell
supernatant
Polyclonal Antibodies
Classic antibodies
A Third Player
Higher affinity
Recognize a greater variety of epitopes that are
immunogenic in mice
Many small compounds or peptides elicit good
immune response only in rabbits
Less need for antigen retrieval
Enzyme label
Commonly used enzyme labels for IPT
Substrate Chromogen
In order to visualize the enzymes labelling the
antibodies with light microscope, enzyme
substrate reactions, which convert colorless
chromogens into visible colored endproducts, is used.:
Peroxidase- hydrogen peroxide- diaminobenzidine
(DAB): BROWN
3-amino-9-ethylcarbazole (AEC): RED
4-chloro-1-naphthol (CN): KOYU
DARK MAV
BLUE
Aq
M ueo
ou u
nt s
HRP
Rinse
Chromogen
Rinse
Counterstain
Rinse
Mounting
Colon
carcinoma
MS-1375 CEA
Rinse
SPECIME
N
Secondary Antibody
Advantages
Even under light microscope we can read the
results
Disadvantages
Risk of nonspecific staining
Necessary to block endogenous peroxidase
enzyme
DAB is carcinogenic
Applications of Immunoperoxidase
staining
Clinical diagnostics, can be used to localized antigens and to
classifying tumors
Localize hormones