the basics of immunohistochemistry
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THE BASICS OF IMMUNOHISTOCHEMI
STRY
PRINCIPLE Antigen-antibody interactions
Antigen
Antibodies produced Antibodies binds antigen
HOW IT IS EMPLOYED? Antigen = Protein of our interest Antibody= we generate
TECHNIQUES BASED ON THIS PRINCIPLE ELISA
FACS
WESTERN BLOTTING
IMMUNOHISTOCHEMISTY
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
INTRODUCTION Immunohistochemistry is the localization of antigens
(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.
IHC takes its name from the roots "immuno," in reference to antibodies used in the
procedure, and "histo," meaning tissue (compare
toimmunocytochemistry).
TYPES OF VISUALISATION Visualising an antibody-antigen interaction can be
accomplished in a number of ways. Chromogenic
an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction
fluorescent Alternatively, the antibody can also be tagged to
a fluorophore, such as fluorescein or rhodamine
WHAT CELLULAR ANTIGENS CAN WE TARGET?
Cytoplasmic Nuclear Cell membrane
APPLICATIONS disease diagnosis drug development and biological research
TYPES OF IHC Direct Indirect
DIRECT METHOD-PRIMARY ANTIBODY ONLY
one step staining method involves a labeled antibody reacting directly with the antigen in
tissue sections. This technique utilizes only one antibody and the procedure is
short and quick. However, it is insensitive due to little signal amplification and
rarely used since the introduction of indirect method.
Goat anti-actin labeled with 594
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
IHC PROTOCOL
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 microtomeMounting onto glass slides that are coated with an adhesive 3-aminopropyltriethoxysilane (APTS) or poly-L-lysine gelatin, egg albumin or Elmer's glue.
Deparaffinization
Rehydration
Antigen retrieval
BlockingPrimary antibody incubatio
nSecondary
antibody incubatio
n
Detection Counterstaining
Mounting &
observation
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).
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 Avidin - biotin
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 usednormal serum, non-fat dry milk, BSA or
gelatin
NON-SPECIFIC STAINING
Before block After block
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
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