Tissue Prep/Staining

Question 1

Fixing

Answer 1

• prevents deterioration, and hardens tissue.
• formalin is most common: reacts with amino acid to stabilize tissue structure, not good for detail
• all contain glacial acetic acid: counters shrinkage

Question 2

Basic Fixing

Answer 2

• used for mitochondrial staining.

- chromatin is dissolved

Question 3

Why must sample be dehydrated?

Answer 3

-will be embedded with paraffin (hydrophobic material)

Question 4

How is a sample dehydrated?

Answer 4

• series of more concentrated ethanol baths

- will destroy neutral fats

Question 5

What happens when a sample is cleared?

Answer 5

-alcohol is replaced with xylene or cedar oil

Question 6

What does clearing do?

Answer 6

Removes the paraffin embedded medium.

Question 7

List three clearing agents.

Answer 7

• xylene
• cedar oil
• carbon tetrachloride

Question 8

What is embedding process?

Answer 8

• specimen moved through three melted paraffin baths.
• placed in mold, filled with melted paraffin; after final bath.
• placed in cold water bath for rapid hardening.

Question 9

How would you prepare thin slice of tissue?

Answer 9

• sectioning: fixed rotary microtome makes slices in fixed distances
• sharp razor and tubular holder will produce similar results

Question 10

Why must we stain tissue?

Answer 10

-generally tissues are colorless

Question 11

Steps involved with staining.

Answer 11

• remove paraffin from the slide mounted section with xylene.
• remove xylene with graded [alcohol] down to water.
• apply stain.
• dehydrate with series of alcohol
• remove alcohol with xylene
• cement slide together.

Question 12

Hematoxylin and eosin

Answer 12

• used to display structural features.

- not much about chemical characteristics of the tissue.

Question 13

Eosin

Answer 13

• stains cytoplasmic components and extracellular

- yellow to pinkish

Question 14

What do orcein and resorcin fuchsin stains reveal?

Answer 14

-elastic material

Question 15

Silver Impregnation

Answer 15

-show reticular fibers and basement membranes

Question 16

Lipids

Answer 16

• sudan will show the lipid.

- preservation requires technique without use of alcohol (dehydration)

Question 17

How do basic dyes cause staining?

Answer 17

• react with anionic groups in tissue.
• phosphate, sulfate, carboxyl.
• higher pH allows more availability for binding.

Question 18

List examples of basic dyes.

Answer 18

• methyl green
• methylene blue
• pyronine G
• toluidine blue

Question 19

How do acid dyes bind?

Answer 19

• form electrostatic linkages with cationic groups. amino groups
• use in sequence to provide different results.

Question 20

Acid dye examples.

Answer 20

• acid fuchsin
• aniline blue
• eosin
• orange g

Question 21

Metachromasia

Answer 21

-dye changes color after reacting with tissue

Question 22

What are histochemical staining techniques used for?

Answer 22

-study chemistry of cells and tissues

Question 23

Schiff reagent reaction

Answer 23

-reacts with aldehyde groups to form a deep-pink color after exposure to HCl

Question 24

Periodic acid-Schiff reaction

Answer 24

-periodic acid cleaves bonds of carbohydrates to form aldehyde group

Question 25

How can RNA organelles be stained?

Answer 25

- use of basic dyes | - require control slides to note basophilic substances

Question 26

Immunocytochemical techniques are used for?

Answer 26

-detect the presence of antigens by monoclonal antibodies

Question 27

Antigens

Answer 27

- proteins - glycoproteins - proteoglycans

Question 28

Antibodies

Answer 28

-activated B cells form monoclonal antibodies ( specific)

Question 29

How are antibodies stained?

Answer 29

- fluorescent dye - conjugated with visible substance for light microscopy - gold or ferritin to produce visible marker in TEM

Question 30

What is indirect labeling?

Answer 30

-marker attached to second antibody, specific to antibody which located the antigen of interest.

Question 31

Steps taken in order to visualize tissue on a slide.

Answer 31

- fixing - dehydration - alcohol removal - embedding

Question 32

What is the process of staining?

Answer 32

1) remove paraffin with xylene. 2) remove xylene with gradient [alcohol] 3) apply stain and dehydrate with [alcohol] 4) clear alcohol with xylene 5) cement slide to prevent changes

Question 33

What is a hybridoma?

Answer 33

-Fusion of activated B cell and myeloma.

Question 34

Acid Fixative

Answer 34

-fix chromatin, nucleoli, spindle fibers

Question 35

Carnoy's Fluid

Answer 35

Acid fixative. alcohol, chloroform, glacial acetic acid. Preserves glycogen.

Question 36

Zenker's Fluid

Answer 36

Acidic. potassium dichromate, mercuric chloride, glacial acetic acid. Used for high detail.

Question 37

Bouin's Fluid

Answer 37

picric acid, formalin, glacial aceitc acid. Prolonged washing process.

Question 38

Zirkle-Erliki Fixative

Answer 38

potassium dichromate, ammonium dichromate, copper sulfate, distilled water.