IMMUNOHISTOCHEMISTRY

ON PROTEIN EXPRESSION
DETECTION
(Moleculer Technology in Moleculer Research)
By
dr. H Agung Putra, Msi. Med

The Chief of Stem Cell and Cancer Group Study

(SCCR)
 IMMUNOHISTOCHEMISTRY
Definition:
is a method of protein staining used to:
Define the presence of specific protein inside celluler
(location)
– Membrane
– Cytoplasm
– Nucleus
– Intarcelluler space
By employing
– labelling antibody as specific reagen (reaksi imunologi)
– Enzymatic reaction for visualizing target protein as
marker.
IMMUNOHISTOCHEMISTRY

Immunologic Enzimatic
Reaction Reaction
IMMUNOCYTOCHEMISTRY

Sampel derived from:

–Cell culture
–Cell suspense
–Cytospins
Not from tissue
 Imunocytochemistry versus
Immunocytochmistry
• IMMUNOCYTOCHEMISTRY
– Sample: cell culture
– Targe tcell fixation : coverslip
– Do not need “Retrieval antigen process”

• IMMUNOHISTOCHEMISTRY
– Sample : “ Blok paraffin atau FS”
– Tissue fixation : glass slide (covered by Poly L Lisin)
– Deparaffinization and Dehidrazation process
– Need “Retrieval antigen process”
Specific protein intracelluler
• What’s that ??
• How they can get inside
celluler ?
• Is that a normal or patologic ?
 Spesific Protein Detection
• The Product of Gene Expression
• Important for molekuler analysis:
Qualitative
Gene Expression
The Strukture of Gene

Amino Acid
 The Site of Protein
Location:
– Membrane
– Cytoplasm Celluler
– Nucleus Fungsional State
– Intracelluler space
Normal
 Functional protein types
Aktivasi
Mutation Inaktivasi Epigenet
Amplikasi
 Detection of Celluler Protein
• the presence/ situs of spesific
protein inside celluler – tissue
Determining for celluler
functional state:
“ Signaling pathway “
 Celluler functional
The gene expression is
producted by:
1. The cell-tissue natural mechnism
2. The cell-tissue treatment: Research-
Medical therapy
The Advantage of Knowing for
Gene Expression
Valuable information for:
1. Clinical doctor in hospital :
a) Diagnosis accuration
b) Prognosis prediction
c) Target therapy
2. Researcher
The moleculer analysis and eksploration
for several phenomena that occur at
celluler level.
 Natural Mechanism:
Tyrosine kinase Signaling Pathway
 Patologic Gene Expression
Point
Mutation
transition
transversion

Silent mutation

Missence mutation

Non sense mutation

Mutation of chromosome
Translocation of chromosome

Overekspresion of ErBB2 gen

 The Principal of
Gene Expression Detection
• Imunology Concept
“ The ability of antibody in binding for
specific sequence of amino acid ----
epitope”
• Immunohystochemistry : qualitative
 Immunology Concept
The basic of moleculer Technique
Immun System
“ The developed complex defense system of
body in an effort to protect the body
toward a lot of antigen invasion,
By producting either anormous celluler or
moleculer that spesific capabilities in
recognizing ang eliminating the various of
antigent.”
Imunity provide the celluler and moleculer protein
to antigen, through 2 system
1. Non specific immun, “innate immunity”
2. Specific immun , “adaptive immunity.”

 Antigen: an object that can trigger the imun

respon

 Antobody: body respons

 ANTIGEN and EPITOPE

Antigen is chemical substance derived from

organism (microba, virus, fungus, parasite),
food material and molecul product of body
metabolism that can be recognized and tightly
binded either by celluler or moleculer imunity.

Imunity celluler or Antobody can only recognize

the at discreate site of antigen : ----- EPITOPE
 Epitope
• An active regio immunogen, that can bind the
specific membrane receptor of antibody or
lymphocytes as antigenic determinants.
• The respon antibody’s just to epitope

• The strukture epitopes of antigen’re diverse -

---- the responsive to different antibody
Epitope-antibody
 The Principal binding of Ab- Ab
Antibody Antigen

Nukleus

Access

 Preparation of sample
 Fixation
 Permaeablition
Antibody monoclonal
Antibody monoclonal product
Direct-indirect method of IHC
Sistematika IHC-ICC
 “Heat Induce Epitope Retrieval (HIER) ”

Formalin Moleculer cross-link

Change of
Fixation epitope
structure

Alkohol Breaking of H20 banding

Slide

Heat solution
Dr. Shan-Rong Shi
IHC Process
 THE INTERPRESTATION OF
IMMUNOHYSTOCHMISTRY
“Staining Patterns”

“agreement of significance.”
a
b c b a c
• a. Sitoplasma
• b. Nuklues
• c. membran
 SCORING ASSESMENT OF IMMUNO
 The German semi-quantitative scoring system
Semi-kuantitatif --- 2 Component:
1. Intensity cell staining
2. Prosentase cells staining
•0 : no staining
• +1 : weak staining,
• +2 : distinct staining,
• +3 : very strong staining.
• Nuklear staining
• Cytoplasmic staining (Nakano, 2008
 HSCORE = Σ (I x PC),
I : intensity
PC: cell staining prosentase
 Caspase-3 expression (ICC)
NEGATIVE EXPRESSION POSITIVE EXPRESSION
Caspase-3 Expression (ICC)
Apototic bodies
 THANKS YOU
WASSALAMUALAIKUM