WEEK 11: Immunohistochemistry in histopathology Flashcards
what is immmunohistochemistry?
Immunohistochemistry is a method used for localising specific antigens in both tissues and cells based on antibody-antigen recognition
what retrieval method increased the intensity of IHC?
heating at high temps - antigen retreival
what is an antibody?
An ANTIBODY has the property of binding with a second molecule SPECIFICALLY called an ANTIGEN.
what is an epitope?
The epitope is a cluster of Amino Acid residues on the antigen that will bind specifically to an antibody.
what are polyclonal antibodies?
what is background staining? How is it caused?
Poor staining result
This is caused by :
Non-specific antibody/antigen binding
Endogenous enzymes
Peroxidase activity
Endogenous biotin
what is the direct method?
antibody joins to antigen and there is a chromogen (coloured substance) attached
avidin-biotin method?
primary antibody is joined by secondary antibody that has the avidin-biotin complex (colour)
polymer kits.
primary then secondary antibody, which is attached to djshafjkashfjkhsaf
how is tissue fixed? why is it the most critical step?
1:10 ratio of tissue to formalin.
Fixation optimises preservation but can also affect immunohistochemistry results.
2 types of fixative
Coagulant fixatives e.g alcohol based
Cross linking fixatives e.g formaldehyde
what is the longest time to leave fixative on tissue? What will happen if its left for too long?
48 hours
Over-fixation can result in staining issues due to strong cross linking of tissue proteins making antigenic sites undetectable by the process
adv of formalin fixative
Formalin fixation has many advantages:-
Good morphological preservation
Economical
Good antigen preservation
For antigens that are modified by fixation, effective Antigen Retrieval will recover them, but this has to be precise and effective.
what is the optimal dilution of an antibody?
defined as the dilution at which desired positive staining is achieved and unwanted background staining is reduced to a minimum.
how is a panel used?
to determine origin of tumour. Once identified, it can be further subtyped with another panel
