Lecture 1

Question 1

what tissue preparation method retains lipids and antigens?

Answer 1

frozen preparation

Question 2

what tissue preparation loses lipids and some antigens?

Answer 2

paraffin

Question 3

what are the benefits of paraffin preparation?

Answer 3

good for long term storage, easy to section, thin sections (5micrometers)

Question 4

downfalls of paraffin preparation

Answer 4

loses lipids and antigens, harsh treatment, is time consuming

Question 5

benefits of frozen prep

Answer 5

fast, retains lipids and antigens, better for some antibody labeling

Question 6

downfalls of frozen prep

Answer 6

not good for long term storage, thicker sections (10-15 micrometers), difficulty to section

Question 7

benefit of electron microscopy prep?

Answer 7

able to view ultrastructures

Question 8

downfall of electron microscopy prep?

Answer 8

difficult and time consuming

Question 9

general steps for slide preparation

Answer 9

1. tissue removed
2. dissected into small pieces
3. fixed in formalin
4. dehydrated in ethanol
5. cleaned in xylene
6. infiltrated with xylene/paraffin
7. embedded in paraffin
8. cut into thin sections using a microtome (5-6micrometers)
9. mounted on slides
10. stained

Question 10

general steps of frozen section prep

Answer 10

rapidly fixed and frozen

cut using cryostat

Question 11

when is frozen sectioning commonly used?

Answer 11

during surgery to make rapid dx

Question 12

what is considered semi-thin sectioning?

Answer 12

0.5-2micrometer

Question 13

what is considered ultra-thin sectioning?

Answer 13

30-60 nanometers

Question 14

how are semi-thin and ultra-thin sections usually embedded?

Answer 14

expoxy, plastic, acrylic resin

Question 15

after tissue specimens are mounted, what must happen in order for water soluble slides to penetrate the sections?

Answer 15

embedding process must be reversed, remove the paraffin (xylene, alcohol, water)

Question 16

Hematoxylin color, affinity, and examples

Answer 16

toluidine blue, methylene blue - stains acidic structures purple

• basophilic tissue with ph<7
  ex. nucleus, DNA, RNA, anionic macromolecules

Question 17

Eosin color, affinity, examples

Answer 17

acid fuchsin (pink) - acidic dye, stains basic structures pink 
acidophilic tissue with ph>7
ex. cytoplasmic, membrane proteins

Question 18

What are the lipid soluble dyes and their affinities

Answer 18

Sudan black, oil red O
long chain hydrocarbons
ex. fats and oils, lipids in the liver

Question 19

Example of multicomponent dyes

Answer 19

Periodic Acid-schiff (PAS) (intense magenta)

trichrome (Masson)
collagen=blue
nuclei=black
cytoplasm=pink

Question 20

what are multicomponent dye affinities?

Answer 20

complex carbohydrates

ex. glycogen, glycosaminoglycans

Question 21

Most common stain

Answer 21

hematoxylin and eosin (H&E)

Question 22

What color do basophilic tissues stain? are these tissues basic or acidic?

Answer 22

purple, acidic

Question 23

what color do eosinophilic/acidophilic tissues stain? are these structures basic or acidic?

Answer 23

pink, basic

Question 24

what is the purpose of staining

Answer 24

allows better visualization of cellular and extracellular components

Question 25

what is the purpose of embedding

Answer 25

reinforces tissue with harder supporting matrix, making it easier to section

Question 26

what is the purpose of fixation?

Answer 26

preserves the specimen and prevents putrefaction and autolysis of tissue

Question 27

what is the purpose of dehydration?

Answer 27

removes water from tissue to allow for paraffin embedding

Question 28

what is the purpose of clearing the tissue>

Answer 28

placing the specimen in solution that is miscible with water and paraffin (clean)

Question 29

what is the resolution of the human eye?

Answer 29

200 μm

Question 30

what is resolution of the light microscope

Answer 30

0.2 μm

Question 31

what is the resolution of the electron microscope?

Answer 31

0.002 μm

Question 32

what structures cannot be resolved by a light microscope?

Answer 32

ribosomes, membranes, actin

Question 33

What other types of microscopy are used to visualize living, unstained cells?

Answer 33

phase contrast | differential interference

Question 34

characteristic of differential interference microscopy?

Answer 34

image is more 3 dimensional, used to visualize living unstained cells

Question 35

what method is known for taking very sharp images?

Answer 35

confocal laser scanning microscopy

Question 36

what is characteristic of phase contrast microscopy?

Answer 36

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

Question 37

Method of collection of confocal laser scanning?

Answer 37

collects light from very thin focal plane, eliminates background, makes sharp image

Question 38

How is fluorescence microscopy achieved?

Answer 38

slides are irradiated with UV light of proper wavelength and the emission of light and color is in the visible portion of the spectrum

Question 39

what does acridine orange bind to?

Answer 39

nuclear DNA - yellow | RNA rich cytoplasm - reddish orange

Question 40

what is prussian blue used to stain?

Answer 40

iron

Question 41

how are phosphatase, dehydrogenase, and peroxidases tagged?

Answer 41

chemical reactions that produce a color change or precipitate

Question 42

how does immunohistochemistry work?

Answer 42

specific proteins are tagged by utilizing interactions between labeled (with fluorescent compound or peroxidase or alkaline phosphatase) antibodies and their antigens within cells

Question 43

what does transmission electron microscopy (TEM) produce

Answer 43

cross sections of cells showing very small organelle structures that are NOT visible with light microscopy HELPFUL IN STUDYING MEMBRANE STRUCTURE AND ORGANELLES

Question 44

what does scanning electron microscopy produce?

Answer 44

detailed 3-D surface features of cells and tissues

Question 45

what are examples of artifacts?

Answer 45

folds, tears, bubbles, cell shrinkage, precipitate (clumbs of dye or dust)