Histology and virtual microscopy Flashcards
How do you prepare specimens for light microscopy?
1) Stabilise structures by chemical fixation
2) Dehydrate and infiltrate tissues with paraffin or plastic
3) Embed tissues into paraffin or plastic block
4) Cut slices 3-10 micrometers thick, collect sections on slides
5) Rehydrate and stain with haemotoxylin (stains basophilic structures)
6) Counter stain with eosin (stains acidophilic structures)
How do you prepare specimens for electron microscopy?
1) Tissues fixed with glutaraldehyde (cross-link protein) and osmium tetroxide (cross-links lipids, also an electron dense stain)
2) Dehydrate and infiltrate tissues with plastic
3) Embed and block fixed tissues in plastic
4) Cut tissues 50nm thick and collect on slides
5) Stain sections with heavy metal salts (lead citrate and uranyl acetate) which bind proteins and nucleic acids
6) Viscualise in TEM; heavy metal stains block electrons to create contrast
Which stain is used to prepare slides for light microscopy?
H and E
Which stain is used to prepare slides for electron microscopy?
Heavy metal salts, e.g. lead citrate and uranyl acetate
Principles of light microscopy
2D image
0.2 micrometre resolution
Cell features more visible with staining
Process of light microscopy
Light shines through lens to specimen
Shines through objective lens through eyepiece lens to eye
Multiply lense to give magnification
Resolution based on wavelength
What are the principles of electron microscopy?
Most images are TEM (electrons pass through instead of light) Check scale bar Images are 2D and black and white Resolution of 0.2 nanometers Stain with electron dense metal stain
What three things would you use to interpret slides?
Morphology, location and function
What is a nucleus with a crypt base?
Round, typical of epithelial cell
What does a nucleus in smooth muscle look like?
Elongated spindle shape, found in centre of cell
What are the four stains we use?
H and E
Trichrome
PAS
IHC
What is H and E dye?
Most common stain in biology
Hematoxylin - basic dye, stains acidic structures purple
Eosin - acid dye, stains basic structures pink
What is trichrome dye?
Also known as Masson triple stain Used to stain matrix components Mixture of dyes Recognised by green/blue staining Blue/green is mucus/collagen Red is cytoplasm
What is PAS stain?
Periodic Acid Schiff
Detects sugar groups
Stains a red colour
Basement membranes - glycocalyx
What is IHC dye?
Immunohistochemistry
Detects proteins
Uses an antibody linked to an indicator usually on an enzyme that will produce a colour visible by light microscopy
