Test 2. Lab 10 Flashcards
*define immunohistochemistry
combines hisological, immunological and biochemical techniques for the identification of specific tissue components by means of a specific antigen/antibody reaction tagged with a visible layer.
AKA
It combines histological immunological and biochemical techniques to identify specific components
IHC is an application of __________ to tissue preparation for the___________ of target antigens:
Wide range of specific antibodies
Highly sensitive detection system
antibodies
localization
what is the principle of immunohistochemistry?
The principle of immunohistochemistry is to LOCALIZE ANTIGENS IN TISSUE SECTIONS by the use of labeled antibodies as specific reagents through antigen-antibody interactions that are VISUALIZED by a marker such as fluorescent dye, enzyme, radioactive element or colloidal gold.
- how can you localize antigens in tissue specimens (immunohistochemistry)
by the use of labeled antibodies as specific reagents through antigen-antibody interactions
How can you visualized antigen antibody? aka what markers can you use?
fluorescent dye, enzyme, radioactive element or colloidal gold
“antibodies”
__________ that are produced by plasma cells and used by the immune system to identify and neutralise foreign objects, ie. bacteria and viruses
Glycoprotein
• Recognise a specific Antigen- mainly_________, __________, ______-
proteins, glycoprotein, polysaccharides
*What are two different types of antibodies? and name what makes them different?
Polyclonal antibodies: Large complex antigens may HAVE MULTIPLE EPITOPES and elicit several antibody types. Mixtures of different antibodies to a single antigen are called polyclonal antibodies.
Monoclonal antibodies: Antibodies SPECIFIC FOR A SINGLE EPITOPE and produced by a single clone are called monoclonal antibodies and are commonly raised in mice.
- Labeling Antibodies:
Antibodies are not visible with standard microscopy and must be labeled in a manner that does not interfere with their binding specificity.
Common labels include ____________ (eg, fluorescein, rhodamine), ____________- demonstrable via enzyme histochemical techniques (eg, peroxidase, alkaline phosphatase), and _________________- for use in electron microscopy (eg, ferritin, colloidal gold).
fluorochromes
enzymes
electron-scattering compounds
*name 3 common labels used to label antibodies. Give example in each subgroup
fluorochromes- fluorescein, rhodamine
enzymes-peroxidase, alkaline phosphatase
electron-scattering compounds- ferritin, colloidal gold
*name the two methods for antigen binding
direct method
indirect method
- define direct method and two step indirect method for antigen binding?
direct method: one antibody label binds to antigen
two-step indirect method- 2 antibody- 1 primary binds to tissue and the second antibody binds to primary antibody
What are some applications where antigen detection can be used
Cancer diagnostics
differential diagnosis
Treatment of cancer
Research
- what are the steps involved in the general immunohistochemistry protocol?
- Fixation: 4% PFA
- Permeabilize: Triton X
- Blocking of nonspecific sites : 10 % Fetal Bovine serum
- Primary Antibody staining (Ki 67 Ab)
- Secondary Antibody staining (Fluoroscence Labelled)
what is the Ki 67 antibody
Ki67 antigen is the prototypic cell cycle related nuclear protein, expressed by proliferating cells in all phases of the active cell cycle (G1, S, G2 and M phase).
It is absent in resting (G0) cells. Ki67 antibodies are useful in establishing the cell growing fraction in neoplasms
quantified by determining the number of Ki67 positive cells among the total number of resting cells = Ki67 index
