Immunohistochemistry - Week 5 Flashcards
what is the definition of immunochemistry
proteins studied in situ within cell/tissues using labelled antibodies
what is the definition of immunocytochemistry and immunohistochemistry
immunocytochemistry:
the study of the proteins within cells (variable or fixed)
immunohistochemistry:
the study of proteins within tissues (fixed or frozed)
what is the application of immunochemistry
applications:
- cancer diagnosis
> cell markers for proliferation and apoptosis
- Research
> Location(s) is important to study function
what are the 3 different types of label and there use
Labels:
- Fluorescent dyes
> used in immunofluorescence, the dye fluoresces under uV when chemically bound to primary and secondary antibody
- Enzymes
> enzymes converts soluble substrate to insoluble product in order to localise the target protein. examples, horseradish peroxidase and alkaline phosphatase. - Colloidal gold
> used with light and electron microscopy
why is the washing steps important
it is important to remove unbound antibodies in order to get accurate results.
what is fluorescence
an energy emission phenomenon seen in some molecules
how do molecules that are promoted to higher energy levels by EM radiation lose energy
molecules promoted to higher energy levels by EM radiation lose energy by:
- EM radiation emitted by the electrons that are transitioning to lower energy levels
- Heat loss due to bond vibration/rotation
what are 2 key facts about molecules with aromatic ring structures
Molecules with aromatic ring structures:
- Low bond vibration/rotation
→ their excess energy is lost as fluorescent light.
what is the purpose of fluorescent dyes or probes
they are used so you can visualise biological components
what are the different things that can be visualised using fluorescent dyes or probes
- Cell surface receptors.
- Intracellular organelles.
- Nucleic acids.
- Apoptosis markers.
what are 3 examples of fluorescent dyes that are used
Examples of different types of dyes:
- FITC (Fluorescein isothiocyanate). Abs = 488 nm;
Ems = 518 nm.
- Alexa Fluor® 488.
- DAPI (4’,6-diamidino-2-phenylindole). Used to stain nucleic acids.
why are fluorescent proteins used in microscopy
In living cells, fluorescent proteins are most commonly utilized to track the localization and dynamics of proteins, organelles, and other cellular compartments.
what are 2 examples of fluorescent proteins
Examples of fluorescent proteins:
GFP (Green fluorescent protein). Emits green light.
PE (Phycoerythrin). Emits red light.
how are samples prepared in immunohistochemistry
Fixation
🡥 🡦
Tissue Sectioning 🡢 Section before staining
sample 🡥
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Freezing
how is the tissue sample prepared in 6 steps from start to finish in immunohistochemistry
Tissue Sampling: Quickly after removal from organism to prevent cellular/tissue degradation.
Fixing: Immobilises , kills & preserves cells/tissue.
> Permeabilises cells to staining reagents.
> Cross-links macromolecules – stabilised & fixed in
position.
> Organic solvents e.g. methanol: acetone.
> Reactive aldehydes e.g. formaldehyde.
Embedding: Soft tissue requires treatment with liquid resin/wax → hardens tissue for sectioning.
Freezing: Dry ice (−80°C) or liquid nitrogen (−196°C).
Sectioning: The sample is cut into 1 – 10 µm thick sections with a microtome before mounting on a microscope slide.
Staining: Primary antibody to the protein of interest + labelled secondary antibody or conjugated antibody. Labels are either colourimetric or fluorescent.
why are cells permeabilised
in order for intracellular target antigens to be expressed in cytoplasm and/or nucleus the staining antibodies need to transverse cell and/or nuclear membrane thus for the transgression to occur the cells must be permeabilised.
what organic solvents used in fixing also permeabilise cells
examples of organic solvents for fixing that also permeabilise are:
- methanol
- acetone
what are detergents used to permeabilise cells
- Triton X-100
- Tween 20
what are incubation times strictly monitored when cells are being permeabilised
incubation times are strictly monitored because over-exposure can lead to cell destruction
how do reagents create holes when cells are being permeabilised
reagents create holes in membranes by dissolving the phospholipid layer
why is blocking used in immunochemistry
blocking is used to prevent non-specific binding of antibodies (except to target antigen) to proteins.
how does blocking work in immunochemistry
A blocking buffer is a solution of irrelevant protein, a mixture of proteins, or other compounds that passively adsorb to all remaining binding surfaces of the plate. this reduces background interference and non-specific binding.
what is commonly used as blocking agents in immunochemistry
- BSA
- casein
- proprietary proteins
what is the main benefit of blocking agents in immunochemistry
blocking agents together with washing steps reduces background staining
example of immunohistochemistry, which protein used, sample used, antibodies used and substrate used
PAX8 protein: Transcription factor over-expressed in epithelial ovarian cancer cells.
Sample:
The epithelial layer of human ovarian carcinoma.
Antibodies:
Primary: rabbit anti-human.
Secondary: anti-rabbit HRP-labelled.
Substrate:
Diaminobenzidine (DAB).
where is L1-CAM and PAX8 coexpressed
L1-CAM and PAX8 are co-expressed in collecting duct cells of normal renal tissue.
why are fluorescent microscopes better than light microscopy or colourimetric staining
Fluorescent microscopes allow the merging of such images which is not possible with colourimetric staining and light microscopy
how is the tissue sample prepared in 5 steps from start to finish in immunocytochemistry
The extracellular matrix surrounding cells has been removed.
Cells can be:
Isolated from a block of tissue.
Grown in cell culture (adherent and suspension cells).
Cell culture: Cells are grown under controlled conditions outside of their natural environment.
Fixing: Immobilises, kills and preserves the cells.
Permeabilises cells to staining reagents.
Cross-links macromolecules – stabilised & fixed in position.
Organic solvents e.g. methanol: acetone.
Reactive aldehydes e.g. formaldehyde.
Staining: Primary antibody to the protein of interest + labelled secondary antibody or conjugated antibody. Labels can be either colourimetric or fluorescent.
