Immunocytochemistry Flashcards
What is the aim/use of immunocytochemistry?
The use of labelled antibodies to bind to antigens in a sample, to stain cellular components in cytological/histological prep. Allows for their visualisation.
What part of the antigen is most considered in this technique?
Epitopes - The parts of the antigen which actually bind to the antibody (at its paratope)
What part of the antibody is most considered in this technique?
Paratopes - the regions of the antibody which bind to antigens (at their epitopes)
What is the difference between monoclonal and polyclonal antibodies in this technique?
Monoclonal antibodies - recognise ONE epitope of an antigen with the same specificity/affinity
Polyclonal antibodies - recognise MORE THAN one epitope. Specificity and affinity varies
What are the two types of fluorescent labels in ICC?
Direct - the label is attached to the first binding antibody
Indirect - label is attached to a secondary antibody, which binds to an antibody already bound to the antigen epitope.
How do enzyme labels work in ICC?
Attached enzymes produce a coloured substrate when bound to target epitope, which can be detected via spectrophotometry. Optical density (OD) correlates with concentration of antigen.
e.g. DAB is converted from a colourless chemical to brown product at antibody binding site
Which chemical structure gives dyes their colour?
Aromatic (benzene) compounds, particularly paraquinoid rings. AKA chromophores.
Due to free electrons, meaning they can change energy levels and emit light (when falling down energy levels)
What are the two main components of a dye?
Chromophore (colour giving) and auxochromes (ionic region which bind to the tissue)
What are acidophiles?
Tissue components with a negative (basic) charge, which are attracted to positively charged acid dyes
e.g. basic amino acids
What are basophiles?
Positively charged (acidic) tissue components that are attracted to negatively charged (basic) dyes
e.g. DNA is a basophile
