A03 Histology Flashcards
What is fixing and why do we do it?
- Usually a chemical fixative is used to crosslink the proteins and then dehydrated (using alcohol).
- Reduces and often stopsbiological activity, at the same time rendering the cells more amenable to staining
What is embedding and why do we do it?
- The specimen is embedded in molten paraffin wax or resin (for thinner sections), and cooled to room temp where the wax/resin medium is a solid.
- The wax provides physical support for subsequent fine sectioning without damage to the tissue.
What is sectioning and why do we do it?
- A microtome is used to cut extremely thin sections of tissue (5 - 8 µm; 0.005mm - 0.008mm). After sectioning the tissue is mounted (placed) on a glass microscope slide.
- Most tissues are too thick to be examined directly in the microscope and must therefore be cut into very thin slices
What is staining and why do we do it?
- Wax/resin is removed and the sample is rehydrated, then stained with general or specific stains.
- help to identify different components
Advantages of freezing sections and at what temp are they frozen?
- quicker than staining
- Preserves proteins in their natural state
- -160 degrees
What colour does the stain Haematoxylin stain the nucleus, cytoplasm and connective tissue
Nucleus - Purple
Cytoplasm - n/a
Connective tissue - n/a
What colour does the stain Eosin stain the nucleus, cytoplasm and connective tissue
Nucleus - Pink
Cytoplasm - Pink
Connective tissue - Pink
What colour does the stain Haematoxylin & Eosin (H&E) stain the nucleus, cytoplasm and connective tissue
Nucleus - Purple
Cytoplasm - Pink
Connective tissue - Pink
What colour does the stain Haematoxylin and van Gieson (HvG) stain the nucleus, cytoplasm and connective tissue
Nucleus - Grey/blue
Cytoplasm - Green/Yellow
Connective tissue - Collagen - Red
What colour does the stain Masson’s trichrome stain the nucleus, cytoplasm and connective tissue
Nucleus - Purple
Cytoplasm - Brick red
Connective tissue - Green or blue
What colour does the stain Elastic van Gieson stain the nucleus, cytoplasm and connective tissue
Nucleus - Grey/blue Cytoplasm - Green/yellow Connective tissue - Collagen - Red Elastic fibres - Black
What is immunohistochemistry
visualise specific proteins within tissues or cells using antibodies, which can precisely target a molecule of interest
What is a fluorescence microscope?
analyse specimens containing fluorescent molecules under ultraviolet light. These may be naturally occurring (autofluorescence) such as vitamin A or some neurotransmitters, or more commonly when fluorescent dyes are used to stain the specimen.
What is a confocal scanning microscope?
uses a laser to scan the illuminated specimen and collect data from a single area at a time. Data is collected onto a computerand displayed on a high resolution monitor.Computer image processing and analysis removes out of focus flare and the result is an image that has been optically sliced to sections of approximately 1 mm. Multiple images at various depths through the specimen can be collected and three-dimensional images generated.
What are SEM and TEM?
use a beam of electrons generated by a cathode rather than a beam of light. The electrons are attracted to an anode, and the specimen is placed between them, in SEM this beam scans across the surface of the specimen, in TEM, it passes through it. The beam passes through a series of electromagnetic lenses just as light passes through glass lenses in an orthodox microscope. The image is viewed on a phosphor screen, and a photographic plate placed above this screen when a permanent record of the image is required. The higher resolution obtainable using these methods means that the specimen must be very thin, and the fixation techniques used the best available.
