IHC Flashcards
process of detecting antigens in cells of a tissue section by a specific antigen/antibody reaction tagged with a visible label
IHC
combines histological, immunological, and biochemical techniques for identifying specific tissue components
IHC
anything that causes an immune response in animals/humans; stimulates production of antibodies in the host
immunogen
protein produced by plasma cells as the result of an immunogen
antibody
protein biomarker used in identifying cells, triggers the productions of Igs
antigen
recovers masked epitopes by breaking aldehyde bonds created by fixation, as well as unmasking antigens
heat induced epitope retrieval/antigen retrieval
large Y shaped protein produced by plasma cells that is used by the immune system to identify and neutralize foreign objects like bacteria and viruses
antibody
antibodies recognize these parts of foreign objects
antigens
classified as primary or secondary reagents
antibody
binds to the primary antibody
secondary antibody
what 5 kinds of cell antigens are targeted?
-cytoplasm
-nuclear
-cell membrane
-lipids
-proteins
Melan-A, cytokeratin 7, cytokeratin AE1/AE3 antigens are these kinds of antigens
cytoplasmic
ER, PR antigens are these kinds of antigens
nuclear
CD20, CD34, E-cadherin, LCA antigens are these kinds of antigens
cell membrane
one stop staining method that involves a labeled antibody directly binding with a specific antigen in a tissue section
direct IHC labeling
IHC staining method used for skin and kidney biopsies
direct IHC labeling
fastest way to get results but with low labeling intensity because the signal is not amplified
direct IHC labeling
uses an unlabeled primary antibody which is attached to the specific antigen; then a labeled secondary antibody is applied and attaches to the primary antibody, allowing visualization
indirect IHC labeling
longer method but less expensive and more sensitive
indirect IHC labeling
PAP method uses this complex with indirect IHC staining
peroxidase anti-peroxidase complex
ABC method uses this complex with indirect IHC staining
avidin-biotin complex
SP method uses this complex with indirect IHC staining
streptavidin peroxidase
secondary antibody is conjugated to several biotin molecules which recruit complexes in these three methods
ABC, SP, PAP
does not rely on biotin, uses a polymer backbone to which multiple antibodies and enzymes can be conjugated; secondary Abs have anti-mouse Ig and anti-rabbit Ig
polymer-based immunohistochemistry
enzyme is used as a label
immunoenzyme method
example of an immunoenzyme method
DAB
fluorochrome is used as a label
immunofluorescence method
example of an immunofluorescence method
FITC
colloidal gold particles are used as a label
immunogold method
this method is used in EM
immunogold method
heterogenous mix of antibodies that recognize several epitopes, derived from different types of immune cells
polyclonal antibodies
specific to one epitope of an antigen, derived from a single cell line
monoclonal antibodies
easier and cheaper to produce antibody
polyclonal antibodies
difficult and more expensive to produce
monoclonal antibodies
duration of time calculated from when tissue is removed from the body to when tissue is placed into fixative
cold ischemic time
false positives are common in the _______ of large tissue blocks or in tissues with _________ fixation
center; delayed
tissue blocks should be no more than _ cm sq or _ mm thick
2; 4
used for identification of carcinomas but may also be expressed in some sarcomas
cytokeratin
used for identification of yolk sac tumors and HCC
alpha fetoprotein (AFP)
used for identifying GIST and mast cell tumors
CD117 (C-KIT)
used for identifying RCC and ALL
CD10 (CALLA)
used in identifying prostate cancer
PSA
used for diagnosing breast and gyn and prognostic for breast cancer and response of therapy
ER/PR
cell proliferation marker
KI67
T cell lymphoma markers (2)
CD3, CD5
B cell lymphoma markers (2)
CD20, CD10
Hodgkins lymphoma markers (2)
CD15, CD30
melanoma panel (2); mesenchymal markers
Mel A, HMB45
breast panel (4)
ER, PR, HER2, androgen receptor
CUP stands for
carcinoma of unknown primary
used to test a protocol or procedure and make sure it works
positive control
used to test the specificity of an antibody involved
negative controls
used to verify the specificity of the antibody and the validation of the protocol chosen
controls
this form of fixation is best for demonstrating good architecture
aldehyde
this form of fixation of is best for demonstrating cell membrane antigens and cytokines
frozen
paraffin loses antigenicity after ____ weeks
2
once sections have been deparaffinized and hydrated, do not let them ____
dry
process of recovering antigenicity of tissue that has been masked by formalin fixation and paraffin embedding
antigen retrieval
sections are soaked in retrieval solution and heated between
95-100 deg celcius
sections are incubated in enzyme solution for 5-30 mins at
37 deg celcius
enzyme antigen retrieval
PIER (protease)
PIER is bad for
morphology
the blocking step decreases
background staining
frequent causes of background staining is endogenous (2)
enzyme and biotin
use normal serum from the secondary host to block this
endogenous biotin
use hydrogen peroxide to block this
endogenous peroxidases
causes of specific background staining (2)
-polyclonal antibodies
-inadequate fixation
causes of nonspecific background staining (3)
-nonimmunologic binding
-endogenous peroxidases
-endogenous biotin
