Tissue Culture and Immunocytochemistry Flashcards
Discuss the conditions required for cells and tissues to thrive outside of the body Describe the fundamental techniques enabling cell culture, including equipment and reagents used Critically evaluate the methods used to separate different types of mammalian cells Discuss ways of measuring cell survival, proliferation, and apoptosis
What is the difference between immunocytochemistry and immunohistochemistry?
Immunocytochemistry is cultured cells being stained, whereas immunohistochemistry is tissue sections being stained
Why are histological dyes important?
Cells are transparent and difficult to see by light microscopy, especially their sub-cellular structures
How does enzyme-linked immunoabsorbant assay (ELISA) work?
Antibodies are linked to an enzyme so that the addition of a substrate produces a coloured reaction when the antigen is present. A colorimetric plate reader allows quantitative analysis, with the depth of colour (amount of antibody) equivalent to the amount of antigen present
Name two commonly used fluorophores
Tetramethylrhodamine (TRITC) and fluorescin isothiocyanate (FITC)
What are the two classes of primary antibodies?
Monoclonal (derived from a single B cell clone and recognising a single antigen site) and polyclonal (derived from several B cell clones, recognising the same antigen but different target sites)
What are the advantages of polyclonal antibodies?
Cheaper, quicker, and easier to produce, can be made in different species’ of animal, and more likely to be successful in an unknown application
What are secondary antibodies?
Antibodies that recognise all antibodies raised in a particular species of animal
Name the steps of immunocytochemistry
Fixation, live cells, sectioning, permeabilisation, blocking non-specific binding, and co-immunocytochemistry
What does fixing do?
It cross-links protein at the cell surface, killing the cells and maintaining them at the moment just before the fixing chemical was added
What are the advantages and disadvantages of frozen staining over paraffin staining?
Advantages: can be stored for longer, better survival of many antigens
Disadvantages: cutting may be more challenging, morphology may not be as good
Describe the process of heat-induced epitope retrieval
Slides are put in a microwave, steamer, or pressure cooker for 20 minutes with a citrate or similar buffer
Why is permeabilisation necessary?
To allow access to intracellular antigens
What is blocking and why is it necessary?
Blocking involves incubating cells with PBS containing protein, e.g. foetal calf serum, to avoid the antibody binding to non-specific binding sites. This allows specific binding to the target antigen to be discernable, rather than hidden by non-specific binding
Why is co-immunostaining useful?
It allows the relative distribution of two or more antigens to be determined
What properties must co-immunostaining antibodies have?
Primary antibodies against each antigen must be raised in different animal species, or different isotypes of the same species, Different secondary antibodies must be used that recognise different primary antibodies, and each coupled to a different fluorophore
