the basics of immunohistochemistry. principle anigen (protein of interest) primary antibody...
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The basics of immunohistochemistry
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Principle
Anigen (protein of interest)
Primary antibody
Secondary antibody
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Immunohistochemistry – what’s good about it?Immunohistochemistry – what’s good about it?•Antibodies bind to antigen in specific
manner•Can be used to locate particular cells and
proteins•Can be used to identify cellular events –
e.g.apoptosis
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Introduction
• IHC takes its name from the roots ▫ "immuno," in reference to antibodies used in the
procedure, ▫ and "histo," meaning tissue (compare to
immunocytochemistry).
• Immunohistochemistry is the localization of antigens or proteins in tissue sections ▫ by the use of labeled antibodies as specific reagents ▫ through antigen-antibody interactions ▫ visualized by a marker such as fluorescent dye, enzyme,
or colloidal gold
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TYPES OF DETECTION• Visualising an antibody-antigen interaction can be
accomplished in a number of ways. • Enzymatic staining
▫ an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction
• immunofluorescence▫ Alternatively, the antibody can also be tagged to
a fluorophore, such as fluorescein or rhodamine
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What cellular antigens can we target?
•Cytoplasmic•Nuclear•Cell membrane
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APPLICATIONS
•disease diagnosis •drug development •and biological research
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Types of IHC
•Direct•Indirect
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Direct method-primary antibody only
• one step staining method• involves a labeled antibody reacting directly with the antigen in
tissue sections. • utilizes only one antibody • procedure is short and quick.• However, it is insensitive due to little signal amplification and rarely used
since the introduction of indirect method.
anti-actin labeled with 594
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Indirect method – primary and secondary antibodies• involves an unlabeled primary antibody (first layer) which react with
tissue antigen, • and a labeled secondary antibody (second layer) react with
primary antibody • This method is more sensitive due to signal amplification • economic
Goat anti-actin
Donkey anti-goat labeled with 488
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IMP!!
• Primary antibody▫Should be raised against the antigen of interest▫E.g.
for detecting human antigens- raise antibodies in specie other than humans!
▫HOW? Injecting human MYOSIN protein in an animal
specie e.g rabbit Antibodies will be raised against human antigen in
rabbit These will be called as RABBIT ANTI-HUMAN
MYOSIN ANTIBODY
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•Secondary antibody
▫Detects the Fc portion of the primary▫must be against the antibody of the animal
species in which the primary antibody has been raised)
▫E.g. In our example primary antibody was rasied in
– rabbit Secondary antibody should detect this rabbit
made antibody So it must be an ANTI-RABBIT Must be raised in another specie (other than
humans & rabbits)
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IHC protocol
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Sample preparation (FFPE) formalin fixed paraffin embedded
▫Tissue fixation ▫ To ensure the preservation of tissue architecture and cell morphology, prompt and
adequate fixation is essential▫ the most common fixative is formaldehyde (FF)
▫Embedding ▫ in paraffin ▫ to maintain the natural shape and ▫ architecture of the sample during long-term storage and sectioning for IHC (PE)
▫Sectioning ▫ Into slices as thin as 4-5 μm ▫ with a microtome
▫Mounting▫ onto glass slides that are coated with an adhesive▫ 3-aminopropyltriethoxysilane (APTS) or poly-L-lysine▫ gelatin, egg albumin or Elmer's glue.
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Deparaffinization
Rehydration
Antigen retrieval
Blocking
Primary antibody incubatio
nSecondary antibody incubatio
n
Detection Counterstaining
Mounting &
observation
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Antigen retrieval
•What?▫ Retrieve your antigen for detection by IHC
•Why?▫ Formaldehyde fixation generates methylene bridges ▫ that crosslink proteins in tissue samples; ▫ these bridges can mask antigen presentation and prevent
antibody binding.
•How?▫ to unmask the antibody epitopes,
1. either by heat (heat-induced epitope retrieval; HIER) 2. or enzymatic degradation (proteolytic-induced epitope
retrieval; PIER).
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Blocking Endogenous target activtiy
• What?▫Quenching or masking endogenous forms of
enzymatic proteins (biotin, peroxidases or phosphatases)
• Why?▫When using Enzymatic detection▫To prevent false positive and high background
detection.• How?
▫Hydrogen peroxide – peroxidases▫ levamisole - Alkaline phosphatase
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Blocking non-specific sites• What?
▫Masking sites that are similar to target sites• Why?
▫antibodies may partially or weakly bind to sites on nonspecific proteins that are similar to target
▫nonspecific binding causes high background staining that can mask the detection of the target antigen.
• How?▫Commonly blocking buffers are used▫normal serum, non-fat dry milk, BSA or gelatin
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Non-specific stainingBefore block After block
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Controls
•Postive control▫ to test a protocol or procedure and make sure it works. ▫ It will be ideal to use the tissue that has the expression
of your antigen▫ If the positive control tissue showed negative staining,
the protocol or procedure needs to be checked until a good positive staining is obtained.
•Negative control▫ To test for the specificity of an antibody involved▫ Exclude the primary antibody – no color should be
obtained