Lecture 1 Flashcards
what tissue preparation method retains lipids and antigens?
frozen preparation
what tissue preparation loses lipids and some antigens?
paraffin
what are the benefits of paraffin preparation?
good for long term storage, easy to section, thin sections (5micrometers)
downfalls of paraffin preparation
loses lipids and antigens, harsh treatment, is time consuming
benefits of frozen prep
fast, retains lipids and antigens, better for some antibody labeling
downfalls of frozen prep
not good for long term storage, thicker sections (10-15 micrometers), difficulty to section
benefit of electron microscopy prep?
able to view ultrastructures
downfall of electron microscopy prep?
difficult and time consuming
general steps for slide preparation
- tissue removed
- dissected into small pieces
- fixed in formalin
- dehydrated in ethanol
- cleaned in xylene
- infiltrated with xylene/paraffin
- embedded in paraffin
- cut into thin sections using a microtome (5-6micrometers)
- mounted on slides
- stained
general steps of frozen section prep
rapidly fixed and frozen
cut using cryostat
when is frozen sectioning commonly used?
during surgery to make rapid dx
what is considered semi-thin sectioning?
0.5-2micrometer
what is considered ultra-thin sectioning?
30-60 nanometers
how are semi-thin and ultra-thin sections usually embedded?
expoxy, plastic, acrylic resin
after tissue specimens are mounted, what must happen in order for water soluble slides to penetrate the sections?
embedding process must be reversed, remove the paraffin (xylene, alcohol, water)
Hematoxylin color, affinity, and examples
toluidine blue, methylene blue - stains acidic structures purple
- basophilic tissue with ph<7
ex. nucleus, DNA, RNA, anionic macromolecules
Eosin color, affinity, examples
acid fuchsin (pink) - acidic dye, stains basic structures pink acidophilic tissue with ph>7 ex. cytoplasmic, membrane proteins
What are the lipid soluble dyes and their affinities
Sudan black, oil red O
long chain hydrocarbons
ex. fats and oils, lipids in the liver
Example of multicomponent dyes
Periodic Acid-schiff (PAS) (intense magenta)
trichrome (Masson)
collagen=blue
nuclei=black
cytoplasm=pink
what are multicomponent dye affinities?
complex carbohydrates
ex. glycogen, glycosaminoglycans
Most common stain
hematoxylin and eosin (H&E)
What color do basophilic tissues stain? are these tissues basic or acidic?
purple, acidic
what color do eosinophilic/acidophilic tissues stain? are these structures basic or acidic?
pink, basic
what is the purpose of staining
allows better visualization of cellular and extracellular components
what is the purpose of embedding
reinforces tissue with harder supporting matrix, making it easier to section
what is the purpose of fixation?
preserves the specimen and prevents putrefaction and autolysis of tissue
what is the purpose of dehydration?
removes water from tissue to allow for paraffin embedding
what is the purpose of clearing the tissue>
placing the specimen in solution that is miscible with water and paraffin (clean)
what is the resolution of the human eye?
200 μm
what is resolution of the light microscope
0.2 μm
what is the resolution of the electron microscope?
0.002 μm
what structures cannot be resolved by a light microscope?
ribosomes, membranes, actin
What other types of microscopy are used to visualize living, unstained cells?
phase contrast
differential interference
characteristic of differential interference microscopy?
image is more 3 dimensional, used to visualize living unstained cells
what method is known for taking very sharp images?
confocal laser scanning microscopy
what is characteristic of phase contrast microscopy?
it takes advantage of different refractive indices of cellular and extracellular structures, and no staining is required.
-is common in tissue culture lab due to quick and easy visualization
Method of collection of confocal laser scanning?
collects light from very thin focal plane, eliminates background, makes sharp image
How is fluorescence microscopy achieved?
slides are irradiated with UV light of proper wavelength and the emission of light and color is in the visible portion of the spectrum
what does acridine orange bind to?
nuclear DNA - yellow
RNA rich cytoplasm - reddish orange
what is prussian blue used to stain?
iron
how are phosphatase, dehydrogenase, and peroxidases tagged?
chemical reactions that produce a color change or precipitate
how does immunohistochemistry work?
specific proteins are tagged by utilizing interactions between labeled (with fluorescent compound or peroxidase or alkaline phosphatase) antibodies and their antigens within cells
what does transmission electron microscopy (TEM) produce
cross sections of cells showing very small organelle structures that are NOT visible with light microscopy
HELPFUL IN STUDYING MEMBRANE STRUCTURE AND ORGANELLES
what does scanning electron microscopy produce?
detailed 3-D surface features of cells and tissues
what are examples of artifacts?
folds, tears, bubbles, cell shrinkage, precipitate (clumbs of dye or dust)
