Immunohistochemistry Flashcards
What is immunohistochemostry used for?
Tumour subtyping, metatstasis, lymphocyte identification, prognostic markers, tumour phenotypes
Explain the tissue fixation step
for the cross linking step we use(4%) paraformaldehyde and (1%) glutaraldehyde forms intermolecular bridges
Then add alchol and acetone which dissolves the lipids and dehydrate the cells and percipitate the proteins then embedded in paraffin
WHy tissue fixate?
To preserve the antigens and prevent their leakage, preserve tissue structure and chemical components,prevents cellular breakdown, protects it from bacteria
What are the two IHC methods? Whta’s the difference
Indirect and direct
Indirect has 2 antibodies, the primary one is labelled with peroxidase and then the secondary antibody binds to it and it’s fluorscently labelled, while direct just has one antibody and is labelled with peroxidase
What is direct IHC and indirect used for?
Direct- to see specific cells in tissues or to see different cells
indirect- amplification of sinal
Explain how biotin avidin complex works?
secondary antibody is biotinylated it binds to avidin which gives it a site for multiple biotin peroxidase secondary antibody labelled to bind to. Detected with avidin enzyme
What is a secondary antibody used to detect multiple antigens?
A polymer backbone with multiple peroxidase and secondary antibodies that binds to primary antibody and amplifies the signal
Why is using polymer backbone better than the conventional detection method?
More sesitive, more specifc, easy to detect, less background
What enzymes are usually used and for what substrates?Why would AP be preffered
Horseradish peroxidase binds to DAP(brown) and AEC(red) Alkaline phosphatase(AP) Becuase of the endogenous Aperoxidase present
Outline general steps of IHC
Cut tissue, rehydrate, antigen retrieval, block peroxidase activity for a bit, incubat in primary antibody, incubate with polymer then enzyme substrate,, counterstain with hematoxylin, dehydrate in alcohol
What rae the two types of antibodies used? What’s the difference
Monoclonal and polyclonal
Binds to a specific antibody and is always identical, binds to antigens nonspecifically and differs, respectively
How to use control for IHC? Why are controls important?
Negative control assay without primary antibodies and positive control antibody aganst an antigen not relevant to the study. Positve tissue and negative tissue control
Can create a range of staining to detect weak positive signals
To control for the inter run variability and operator errors and to confrim the final results are attributed to the question they are asking and not other irrelevant factors
What are possible problems that can arise withIHC?
Autolysis Cross reactivity Antibody or concentration not appropriate Antigen loss Non specific binding to tissues Peroxidase endogenous
Antibody validation. Why isn’t positive and negative tissues enough for validation?
Through western blot using lysates known to express the specific antigen and ones not known to express it(negative).
Because there is variablity from factors
How to retrieve the antigens?
Proteolytic digestion(pepsin, proteinase k), microwave energy, heat and pressure, and then buffers(citrate, EDTA)
