Further Histological Techniques Flashcards
What are some pros to pathologic indexing ?
- fairly quick, lends itself to ‘perioperative’ histology
- easy way to add statistically testable values to a largely qualitative field
What are some cons to pathologic indexing?
- relies on highly skilled hisotlogists to interpret slides
- inter-histologist difference in interpretation ahve potential to introduce sibjectivity
Define Immunohistochemistry
- the use of antibodies to visualise specific molecules located in, on or outside a cell
How do we make IHC antibodies ?
- easiest = inoculate an animal with target protein
- providing antigen is non-self to animal, it will produce antibodies against, which will then be purified
- this produces a range of Igs against different antigens
Define polyclonal antibodies
- mixture of Igs made by inoculating animals & contain reactivity against multiple epitopes
Describe the procedure for animal inoculation
- inoculate mammal with antigen
- wait 28 days, often 3/4 repeated inoculations
- collect blood, separate serum & isolate antibody
What are the 2 ways we can produce IHC antibodies?
- Animal inoculation
- Cell lines
What are hybridoma cells?
- hybridoma cells are an engineered cell made by fusing antibody producing B -cell from an animal, with a type of cancer called myeloma cell
Describe how we produce IHC antibodies from cell lines
- hybridoma cell takes the antibody producing properties from the B-cell, with immortality of cancer cells
- any single hybridoma cell is generated from a single B cell, they produce a single antibody towards epitope of an antigen
Describe the procedure of producing monoclonal antibodies
- inoculate animal with antigen (mice typically)
- remove spleen & isolate B cells
- fuse B-cell with myeloma cell to produce a hybridoma cells
- grow hybridoma cells in culture & harvest antibody from their culture medium
What are antiboides made from hybridoma cells called ?
Monoclonal antibodies
Briefly describe monoclonal antibodies
- antiboyd from single parent clone
- antibody from 1 clone detects 1 epitope on antigen
- usually produced in lab using hybridoma tech.
Briefly describe polyclonal antibodies
- antibodies arsie from multiple B cell clones
- mixture of antibodies that detect different epitopes on antigens
- typical in serum response antigen
What are 2 ways we visualise antibodies on cells ?
- fluorescence
- visible colour change
Define fluorescence as a way to visualise antibodies
- antibodies are conjugated to a fluorescent reporter molecule which can be visulalised using fluoroscence microscopy
What are the 2 types of Antigen retrieval ?
- heat induced antigen retrieval
- enzymatic antigen retrieval
Why is antigen retreival necessary ?
- methylene bridges formed during formalin fixing cross-link proteins, which can mask antigenic sites/epitopes
- therefore antigens need to be retreived to allow antibodies to bind
Briefly describe the physics of fluorescence
- electrons in fluorophore atoms exist in a ground state
-. when energy of a particular wavelength hits it excites electrons to a higher state - eventually electrons return to ground state & release energy they received
What 2 values are associated with fluorophores ?
- excitation = wavelength it absorbs
- emission = wavelength it emits
Why is a secondary antibody used with a fluorophore ?
- increases fluorescence signal from target as multiple secondary antibodies will bind a single primary antibody
- a secondary antibody is an antibody against Igs from species
Why are isotopes important to molecular biologists ?
- using antibodies to react with each other to increase specificity or signal
- need to know that shape of antibodies being used to ensure interactions work
What is a secondary antibody?
- sometimes in IHC a fluorescent marker on an additional antibody which binds to the Fc region of the initial (primary) antibody
What is a benefit of using different antibodies to help visualise antibodies on cells?
- if we use antibodies targeted against multiple different substances or cells & conjugate them to different fluorescent molecules
- we can visualise several cells/structures in the same sample without compromising specificity
What is necessary when multiplexing?
- making sure the primary antibodies are conjugated to different fluorochromes, otherwise different markers will appear to same colour
Describe Nuclear Stains
- nuclear stains in IHC are often DNA intercalating dyes with fluorescence properties
- commonly used stain is DAPI
Describe Phalloidin
- bicyclic peptide which originally isolated from death cap mushrooms
- binds to filamentous actin in a highly selective manner
- can be conjugated to a fluorophore in the same way as an antibody & used as a counter stain to highlight actin fibres
Describe how IHC uses a colorimetric approach
- approach produces a local change in colour on a cell which is visible with conventional transmitted light microscopy
- enzyme is conjugated to a fluorophore rather than an antibody
- the enzymes are treated with a substrate which the enzyme converts into a coloured product at the site of antibody attachment
What enzymes are typically used in IHC colorimetric approach?
- horseradish peroxidase
- alkaline phosphatase
