immunohistochemistry Flashcards
what is immunohistochemistry
method for detecting specific antigens on cells using the antigen/antibody reaction
when is IHC performed
- after routine stains are views
- patient already has diagnosis or relevant clinical history
what does IHC used for
- detects pathological antigens
- can assign lineage to cells/tumors
- determine stage and grade of cancers
- personalized/ precision medicine treatments
- allows us to see distribution and localization of antigens within the tissue
what does IHC detect
many of different antigens
what antibodies can be detected
- polyclonal vs monoclonal
- primary vs secondary
polyclonal is…
injecting antigen to animal to raise an immune response to harvest the antibody
- the Ab is directed against different parts of antigen
monoclonal is..
specific to single part of antigen which raises the immune response
- spleen cells is where the antibody is used in
components of polyclonal
- less expensive
- mixed population of antibodies
- binds to multiple areas of target antigen
- cause cross reactivity less specific
monoclonal components
- very expensive
- single antibody
- binds to specific areas of antigen only
primary
binds specifically to antigen
- primary is labelled = direct IHC
secondary
binds to primary antibody
- when secondary is labelled = indirect IHC
- signal is amplified
how is antigen labelled and detected
- bound to antibody or third layer (enzymes and polymers)
- chromogen added (DAB brown AP red)
- counterstain
why are third layers used
addition specific layers to increase sensitivity and reduce amount of primary and secondary antibody
examples of third layer
PAP
ABC
LSAB
polymer method
PAP
peroxidase anti peroxidase
- third layer antibody binding to second layer
ABC
avidin-biotin complex (3rd layer)
- uses biotinylated secondary antibody
LSAB
labeled streptavidin biotin
- similar to ABC but the complex is not pre assembled
polymer method
dextran polymer can bind up to 70 enzyme molecules along with primary or secondary antibody
what are frozen tissue section used for
- used for immunofluorescence
- fluorophore- linked secondary or primary antibody
- can use multiple labels with the same tissue by using different colour chromophores
true or false: different antibodies need different protocols
true
there can be no delays in…
fixation
routine formalin..
cross links mask antigens
epitope retrieval steps
- heat induces epitope retrieval
- enzyme induces epitope retrieval
microtomy
needs to be skilled
this step needs to be complete
paraffin removal
quenching/ blocking steps
incubate tissue with solutions that bind to non specific sites
quenching/ blocking steps help..
reduce background staining reduce false positive reactions
primary antibodies must be
optimized
what are controls that are used
positive controls
biologically negative controls
reagent negative controls
must be able to contrast with special stain control
positive controls
specimens containing the target molecule
biologically negative controls
specimens that do not contain the target molecule
reagent negative controls
omit the primary antibody
- assess background staining
contrasting with special stain control
single section of biologically positive tissue on the same slide as the patient
