Theory L7 - Biochemical Perspective Flashcards
Why use objective measures?
They can be good for verifying subjective measures
Name some objective methods.
Histology (immunohistochemistry) Enzyme immunoassays (ELISA, EIA, RIA) Protein Analysis (Western Blot) Gene Expression (Northern Blot) DNA Sequence (Southern Blot) PCR RT-PCR
What are the steps in histology?
Fixing - Chemical - to preserve from decay
Processing - dehydrating, clearing out and infiltration -
Embedding - The technique of placing cells or tissue in a supporting medium so that thin sections can be cut.
Sectioning
Staining
- important because the tissue can be easily damaged. Cell membranes are very sensitive to oxygen and can leak/break.
- it is possible to lose what you’re interested in. Artefacts could also be created.
what are examples of histology?
CRESYL VIOLET
LUXOL FAST BLUE
GOLGI STAIN
AMYLOID STAIN
What is cresyl violet?
- identifies neural structure in the brain and spinal cord tissue
- can detect damage and lesions.
- can monitor NEURODEGENERATION in animals.
- dark colour = high density of cells. MORE CELLS = takes up more stain.
CANNOT use on humans, unless POST MORTEM - but not a good choice anyway, because it’s not that specific.
Eg: Chemically lesion animal brain areas, then use this stain to see the extent of damage that was caused - as a manipulation check.
very good in animals.
What is golgi stain used for?
- Stains the ENTIRE NEURON, including dendrites and axon and CONNECTIONS between neurons can be seen - eg. uniploar, bi polar.
- complex networking structure can be investigated
- not good for detecting cell densities, bc not ALL neurons will stain
eg. detecting the effects of childhood neglect on neural connections, and growth of dendrites…
What is Luxol Fast Blue used for?
- stains Myelin
can look at enviro influences on myelin
What is amyloid stain used for?
detects amyloid deposits in the tissue.
- Alzheimer’s
- this stain isn’t specific for alzheimer’s disease
- amyloid is in extracellular space for AD
What is immunohistochemistry?
- Use if you know exactly what causes a condition - what specific PROTEIN you’re looking for.
- very SPECIFIC method.
- can combine with other stains.
- can look for AMYLOID PROTEIN
- is good because you can know that it’s not due to artefacts.
- Cannot let any of the proteins denature!! - use very GENTLE tissue preparation.
- If you let it denature, you will prevent the ANTIBODIES from binding to the ANTIGENS (proteins)
- PRODUCE antibodies that specific bind to your antigen.
- Can use high quality fluroescent dyes.
- very EXPENSIVE and SENSITIVE and SPECIFIC
- can look for multiple proteins at once, just attach different fluorescent markers to each
What is an example for something you’d use immunohistochemistry for?
To investigate Huntington’s disease.
- Look for cell loss - with other stains- can monitor the progression of the condition.
- Then specifically, (Especially in early stages before cell
loss, look for the protein that causes the condition
using immunohistochemistry.
Cell Analysis techniques?
Flow Cytometry Fluorescence microplate assays Cell counting Small animal in vivo imaging Super-resolution microscopy
Why is cell analysis good?
- Cells are the starting point of a myriad of biochemical and physical processes of life
- Can allow us to understand, predict and ultimately influence factors that underlie cell health, proliferation, function and death.
- good, specific, but not in living humans. only post mortem.
SO can’t monitor disease
—> CANCER! - when cells completely lose their identity.
What is small animal in vivo imaging?
- Used to investigate effects of drugs or movement of small molecules in the body of small animals.
- label the drug with flurosence so we can see it move/migrate from injection site and if it crosses BBB
- cannot see the EFFECT of the drug.
- also good to investigate DOSAGE of a drug - see how fast it exists the body.
- expensive machine.
ethics - good for ethics because we can keep an animal for longer. flush the drug do another trial.
What is super-resolution microscopy?
- investigates SYNAPTIC CONNECTIONS, different PROTEINS inside cell body, organelles, nuclear membranes…
- can look at sub-organelle level.
- info about fine structure of cells
eg. mitochondria - site for cellular respiration - problems with mitochondria = neurodegeneration
- must use VERY specific stains
- Pretty much anything can be visualised.
- very expensive type of microscopy.
What are enzyme immunoassays?
- Enzyme immunoassays developed for the QUANTITATIVE determination of proteins (antibodies or antigenes) in a biological sample.
- tissue extract, blood, CSF, Saliva, other Body Fluids.
- originally used to identify antibodies in blood
- need to think about which bio sample you want to use. eg. saliva is the easiest because doesn’t cause stress in participant –> might change levels of cortisol etc
