Lecture 03- Tissue Prep & Staining

Question 1

What are the 4 steps to Tissue Fixing?

Answer 1

1) Fixing 2) Dehydrating 3) Clearing 4) Embedding

Question 2

What is the fixing agent that is most widely used, reacts with amino acids of the tissue proteins, stabilizes tissue structure to prevent deterioration, but is bad for fine cytological details?

Answer 2

Formalin

Question 3

Carnoy’s Fluid, Zenker’s Fluid, and Bouin’s Fluid are what type of fixative?

Answer 3

Acid fixatives. They fix chromatin, nucleoli, and spindle fibers, but not mitochondria or nucleoplasm.

Question 4

Zirkle-Erliki is what type of fixative?

Answer 4

Basic. Fixes mitochondria, but dissolves chromatin.

Question 5

Glutaraldehyde & Osmium Tetroxide are what type of fixative?

Answer 5

Fixative for TEM. They fix proteins and lipids, respectfully.

Question 6

How do you dehydrate samples?

Answer 6

Place tissue in successively increasing strengths of ethanol.

Question 7

What’s the drawback to using alcohol for dehydration?

Answer 7

Ethanol dissolves neutral fats

Question 8

How do you “clear” a tissue sample after it has been fixed and dehydrated?

Answer 8

Remove alcohol and coat tissue with xylene, cedar oil, or tetrachloride.

Question 9

How do you “embed” a tissue sample after it has been fixed, dehydrated, and cleared? (NOT for TEM)

Answer 9

The specimen is moved through several melted paraffin baths, molded, and hardened in a cold water bath.

Question 10

How do you “embed” a tissue sample for TEM?

Answer 10

Infiltrate the tissue with a monomeric resin (epoxy resin) and then polymerize the resin.

Question 11

What are the steps for preparing thin slices of a tissue section for observation?

Answer 11

1) Specimen mold placed in rotary microtome 2) Mold lifted down across sharp blade to cut one thin slice 3) Mold moved forward a pre-determined distance and cut again

Question 12

How do you prepare a section of tissue for TEM?

Answer 12

Same idea as normal, but much smaller scale. Use diamond knives to cut pieces. Float the pieces on to a plastic-coated copper mesh, because they are too fragile. Plastic and copper mesh stay put. Holes in the mesh allow for electrons to pass through.

Question 13

Steps to prepare tissue for staining

Answer 13

1) Remove paraffin w/ xylene 2) remove xylene w/ graded series of alcohol down to water 3) Apply stains 4) Dehydrate by adding alcohol again 5) Remove alcohol w/ xylene 6) Add cement and cover slip

Question 14

Which stain is routinely used to display basic structural features, behaves like both a basic and an acidic dye, stains nuclear material dark blue and purple and extracellular material pink.

Answer 14

Hematoxylin & Eosin (H&E)

Question 15

Which stains are used to specifically reveal elastic material?

Answer 15

Orcein and Resorcin

Question 16

Which stain is used to show reticular fibers and basement membranes?

Answer 16

Silver Impregnation

Question 17

Which stain is fat-soluble and used to demonstrate lipids in a black color?

Answer 17

Sudans

Question 18

Which stains are basic and react to anionic groups (phosphate, sulfate, carboxyl) of tissues?

Answer 18

Methyl Green, Methylene Blue, Pyronine G, Toluidine Blue

Question 19

Which stains are acidic and reacts with cationic groups?

Answer 19

Acid Fuchsin, Aniline blue, Eosin, Orange G

Question 20

What heavy metal stains can be used for TEM?

Answer 20

Osmium Tetroxide, Uranyl Nitrate, Uranyl acetate w/ lead citrate, or platinum and gold for SEM.