Lecture 3 - Tissue Prep And Staining

Question 1

What are the basic steps for fixing tissue and embedding?

Answer 1

1. Fixing
2. Dehydration
3. Removal of Alcohol
4. Embedding

Question 2

What are the benefits and requirements for proper fixing?

Answer 2

• Helps prevent the tissue/sample from further deterioration, and helps harden it for embedding and sectioning.
• All fixatives distort the specimen, but the ideal fixative has the smallest amount of distortion and (with the help of staining) gives the greatest amount of optical contrast

Question 3

Describe formalin

Answer 3

Formalin is one of the most widely used fixing agents.

• When used alone, it’s usually buffered to 10% concentration
• When used with alcohol, it tends to shrink the tissues
• Acetic Acid helps to prevent the effects of alcohol shrinkage
• Though overall useful, formalin isn’t ideal if finer more extreme cytological detail is needed.

Question 4

What are the benefits of using acid fixatives?

What are some types of acid fixatives?

Answer 4

Can help to visualize and fix chromatin, nucleoli, and spindle fibers, but not mitcochondria or nucleoplasm

• Carnoy’s Fluid: Good for preserving glycogen in animal tissues
• Zenker’s Fluid: Allows for sharp histological detail, but must be washed carefully to avoid the formation of black crystals
• Bouin’s Fluid: Allows for Good cytological detail, but requires prolonged and careful washing cycles

Question 5

What are the benefits of basic fixatives?

What is the main basic fixative used?

Answer 5

Basic fixatives can be used to fix tissues where mitochondrial staining is desired, but chromatin is dissolved.
- Zirkle-Erliki fixative: It requires a long fixing time (2 days) and washing under running water.

Question 6

What are the main fixatives for Transmission Electron Microscopy?

Answer 6

• Glutaraldehyde: Preserves proteins by cross-linking them

- Osmium tetroxide: Reacts with lipids (esp. phospholipids) and imparts electron density to cell and tissue structures.

Question 7

What are the benefits to dehydration?

What is the process normally used?

Answer 7

The tissue sample will eventually be embedded and infiltrated with a hydrophobic material (usually paraffin), so all water must be removed from the tissue.

• Dehydration consists of placing the tissue in successively increasing strengths of ethanol until all the water is removed.
• Ethanol dissolves neutral fats and cannot be used for dehydration if it is desirable to leave these intact.
• N-butyl alcohol or acetone may also be used for dehydration.

Question 8

What is the purpose of embedding tissues for staining?

Answer 8

The tissue specimen is moved sequentially through several (usually three) melted paraffin baths.

• After the final bath the specimen is placed in a mold that is then filled with melted paraffin.
• The paraffin mold is rapidly hardened by placing it in a cold water bath.

Question 9

What is usually done to remove alcohol from a tissue prior to embedding?

Answer 9

Xylene, Cedar Oil, or carbon tetrachloride

Question 10

Parafin-block rotary scopes are usually used to section off tissues.
How is sectioning for Transmission Electron Microscopes different?

Answer 10

• Sections are cut at 50 to 150 nm by diamond knives.
• Sections are too fragile for normal handling and must be floated onto a plasticcoated copper mesh grid.
• Plastic is left in place during viewing.
• Holes in the copper grid allow the electron beam to pass through the tissue sample.

Question 11

Why is it preferred that tissues be stained for observation?

Answer 11

Animal tissues are generally colorless. If you want to make out anything, it needs to be stained for more detail.

Question 12

What steps are necessary for parafin-stained specimens?

Answer 12

• Parafin is hydrophobic, which is why tissue needed to be dehydrated first. Doesn’t apply to this, but might be important.
• The parafin needs to be removed by xylene
• After that, the xylene needs to be removed with increasingly weak alcohols, until it’s pretty much water.
• The stains are applied, and the tissue is re-dehyrated with alcohol, which is re-removed with xylene. (Pretty repetitive)
• The process is finalized with a drop of cement (because why not), followed by a cover slip (probably seals the cover)

Question 13

Describe the H&E stain

Answer 13

• Hematoxylin and eosin is a special type of dye that’s useful for detailing structure of the tissue (but not the chemistry)
• Hematoxylin can stain nuclear material and some cytoplasmic components such as Rough ER. Color usually ranges from light blue to dark blue to purple. Note: Hematoxylin is not an basic dye. Though it kind of acts like that
• Eosin is an acidic dye that stains extracellular material a yellowish or pinkish color.

Question 14

What other types of stains are used to reveal what types of structures?

Answer 14

• Orcein and resorcin fuchsin: Reveal elastic material
• Silver impregnation: Shows Reticular Fibers and Basic Membranes
• Sudans: Can identify lipids and fat soluble stains (However, alcohol can’t be used to dehydrate fats)

Question 15

What are the characteristics of basic dyes? What are some examples of basic dyes?

Answer 15

• React with structures heavy in phosphate groups, sulfate groups, and carboxyls. Anything that’s basophilic.
• Examples include Methyl Green, Methylene Blue, Pyronine G, Toluene Blue, Capsacin Magenta
  …nah, that last one I just made up.
• Note, depending on the pH, the color of the stain could look quite different

Question 16

Describe acid dyes

Answer 16

• Bind to electrostatic cationic linkages, such as charged amino bonds.
• Mallory’s Triple Stain uses three different different dyes to stain collagen, cytoplasm, and blood cells different colors.

Question 17

What are more examples of acid dyes?

What does metachromasia refer to?

Answer 17

Acid fuchsin, Aniline blue, Eosin, Orange G

Metachromasia refers to a dye that changes color after reacting to a tissue, such as toluidine blue, which stains cartilage or granules.

Question 18

Define histochemistry

Answer 18

Histochemical techniques can be used to study the chemistry of cells and tissues.
- Example: Perl’s Reaction, Used to demonstrate the iron in tissues, especially in diseases that store iron. Performed by incubating tissues in potassium and HCL. Results are an insoluble blue precipitate.

Question 19

What is the Schiff reaction?

Describe the Fuelgen version.

Answer 19

The Schiff reaction Depicts the formation of aldehydes after being exposed to HCL or periodic acid.

• The Fuelgen version uses HCL to expose aldehyde groups on deoxyribose. Schiff reagent reacts with these aldehydes to form a pinkish color.

Question 20

Describe the PAS version of a schiff base

Answer 20

Stands for Periodic Acid-Schiff Reaction
Periodic acid cleaves bonds between adjacent carbons of carbohydrates and form aldehyde groups. The Schiff reagent then reacts with the aldehyde groups and forms a deep-pinkish color.
PAS-positive substances include Polysaccharides (glycogen), Glycosaminoglycans, Proteoglycans, Glycoproteins, and Glycolipids.

Question 21

Give a clinical application for the PAS reaction

Answer 21

Biopsies of tissues from patients with glycogenoses (glycogen storage diseases)

Clear, Unstained cells imply that they have lost their glycogen content.

Question 22

What is immunocytochemistry?
What are some examples of antigens?
Describe antibodies and give examples.

Answer 22

• Immunocytochemical techniques can be used to study the presence of specific antigens by using monoclonal antibodies
• Proteins, glycoproteins, proteoglycans
• Monoclonal antibodies are derived from activated B cell clones exposed to a specific antigen. These antibodies are very specific. Most antigens have a variety of epitopes (binding sites) that generate a number of different antibodies –polyclonal. A single immune response to an antigen is referred to as monoclonal

Question 23

How are B-Cells used in conjunction with cancer cells in immunochemistry.

Answer 23

• B lymphocytes can mutate into tumor cells resulting in a myeloma. Normal B-cells surrender their ability to replicate
• Myeloma cells acquire the ability to grow indefinitely in culture.
• Fusion of a single activated B cell and a myeloma cell will create a hybridoma that can grow indefinitely in culture and produce a specific monoclonal antibody.

Question 24

What is the difference between direct labeling and indirect labeling?

Answer 24

Direct labeling: Antibodies can be conjugated with a fluorescent dye to produce a visible marker with fluorescent microscopy.
- Antibodies can be conjugated with a visible substance to produce a visible marker for light microscopy.
- Or they can be used with gold or ferritin to produce a marker visible with electron microscopy.
Indirect labeling: In this variation, the marker is attached to a second antibody which is specific to the antibody used to locate the antigen of interest. (Like secondary Imaging?)