Lipids

Question 1

What are some pertinent characteristics of lipids in relation to histotechnology?

Answer 1

Lipids:

1. Structurally heterogenous group of organic substances.
2. Insoluble in water but soluble in alcohol, acetone, chloroform, xylene, and paraffin.

Question 2

What are lipids classified?

Answer 2

Classification of Lipids:

1. Simple Lipids = Hydrophobic in nature
2. Compound Lipids = Hydrophilic in nature
3. Derived Lipids = Hydrophilic in nature

Question 3

Name some examples of simple lipids.

Answer 3

Simple lipids- includes neutral fats, oil and waxes (incl. cholesterol esters). Neutral fats make up 90% of the stored lipids in humans. Triglycerides belong in this group as well.

Question 4

What is an important waxy fat and where is it found?

Answer 4

Cholesterol esters - waxy fat.

Found in adrenal glands (predominately).

Question 5

What do “compound lipids” consist of?

Answer 5

Compound lipids - Consists of glycerol, two fatty acids and usually one fatty acid containing phosphorous or nitrogen.

Question 6

Name some examples of compound lipids. Where can they be found.

Answer 6

Examples are phospholipids (lecithin, cephalins, and sphingomyelin) and glycolipids (cerebrosides and gangliosides). Both found in the Brain and other nervous tissue.

Question 7

What does derived lipids refer to/mean? What are some examples.

Answer 7

Derived lipids- Refer to fatty acid derived from simple and compound lipids by hydrolysis. Steroids are also placed in this class.

Examples are cholesterol, bile salts, sex and adrenocortical hormones.

Question 8

What way are lipid tissues best treated in the histotechnology laboratory when cutting the sections (fixative, etc.)?

Answer 8

Lipid tissues:

Only cryostat frozen section may be used to demonstrate lipid unless the tissue has been fixed in osmium tetroxide.

Question 9

What functions do lipids serve in the body?

Answer 9

o Structural components of cellular membranes
o Storage deposits of metabolic energy
o Protective and insulative properties

Question 10

What colour will Osmium Tetroxide fix lipids to? Why is this fixative not used on a routine basis?

Answer 10

Osmium Tetroxide, will fix lipids to be black seeing not only the outline of the individual fat cells but the inside of it too.

Due to the expense and toxicity of this reagent (can fix your eyes if not careful and working at the fume cabinet), it is usually not used on a routine basis osmium tetroxide. It is used for electron microscopy for lipid-containing material (e.g. cell membrane is rendered electron opaque and is visible).

Question 11

What is a best practice when trying to focus on fatty tissue on the microscope?

Answer 11

Focus on a solid piece of tissue first before going to the adipose area.

Question 12

Will Gordon and Sweets or H&E show adipose cells well?

Answer 12

No, Gordon and Sweets shows the inside of adipose cell to be empty and doesn’t take up most stains well.

H&E will also not show/demonstrate lipid inside adipose cell.

Question 13

What is the principle of stain for Oil Red O?

Answer 13

Oil Red O Principle of Stain:

Selective solubility (physical staining).

Oil Red O is the neutral lipid staining step.

Question 14

What are the main chemicals and their purpose in the Oil Red O technique?

Answer 14

1. Oil Red O –> Stains lipids thru selective solubility (physical staining). This is the neutral lipid staining step.
2. 60% Isopropanol –> Differentiation
3. Harris Hematoxylin (containing Acetic Acid) –> Counterstain step
4. Ammonia water –> Counterstain Enhancement Step

Question 15

What does Oil Red O technique demonstrate? What colours?

Answer 15

Demonstrates neutral lipids in frozen tissue.

Neutral lipids also known as hydrophobic lipids are demonstrated as an orange red color highlighted against a blue background.

Question 16

What type of dye is Oil Red O?

Answer 16

Oil Red O- weak acid dye, red color, attaches by selective solubility.

Dye solvent is either propylene glycol or isopropanol, dye is attracted to lipid by adsorption, and dye dissolves in lipids by selective or preferential solubility.

Question 17

By what principle does Harris Hematoxylin stain the nuclei and cytoplasm of the remaining tissue and what colour?

Answer 17

Harris Hematoxylin

1. Nuclei are counterstained blue with hematoxylin through electrostatic attraction.
2. Cytoplasm will be stained a pale blue color through physical attraction.

Question 18

What does ammonia water do in the Oil Red O technique?

Answer 18

Ammonia Water- This blueing agent merely enhances the color of the hematoxylin stained tissue and increases the contrast between the lipid and background.

Question 19

What would happen if the acetic acid was not in the Harris Hematoxylin in the Oil Red O technique?

Answer 19

If it was Harris Hematoxylin only it would stain reddish brown, but with Acetic Acid in there then it stains blue in one step.

Question 20

What thickness are the frozen sections ideally cut at? Versus what the normal thickness other tissues are cut at?

Answer 20

Frozen sections for Oil Red O: 8-10 um

Whereas normally other tissues are cut at ~ 5um.
(Or I think even thinner to see more detail).

Question 21

Why are lipid tissues typically cut frozen?

Answer 21

Need to use fresh tissue to cut frozen sections because paraffin tissues go through many solutions of alcohol which dissolves the lipids, so to preserve them and be able to stain them fresh tissue is used and sections need to be cut 8-10 microns.

Question 22

Why is a control slide not necessary for this technique?

Answer 22

Because fat can be seen in most tissue, a control is not necessary.

Question 23

After the solution of Oil Red O has been made it should be ______ and then covered? Why?

Answer 23

filtered

Closed container and filtering prevents evaporation of solvent and formation of precipitate occurring in solution.

Question 24

How long does the Oil Red O solution last after being made?

Answer 24

Short shelf life –> Use within 2 hours.

Question 25

Do you do dehydrate, clear and mount as with other tissues (like connective tissue for example)? What type of mounting media is used?

Answer 25

No, Because alcohols dissolve lipids doing DCM is strongly discouraged.

An aqueous mounting media is recommended to preserve lipids.

Question 26

Why should you not press firmly on the coverslip when preparing lipid slides (say to remove a bubble)? What can you do instead?

Answer 26

Adding pressure to coverslip will displace lipids throughout tissue not giving an accurate representation on where lipids are in the tissue.

If there is a lot of bubbles after Coverslipping was done, It is recommended to remove coverslip carefully in warm water and to re-coverslip.

Question 27

Does one orange/red structure or dot indicate a tissue is positive for lipids?

Answer 27

Could be stain precipitate as in the example picture in class.

To be positive we would need to see a lot of orange stained lipids on the tissue.

When staining for lipids there should be a good amount of lipids present.

Question 28

If a tissue can’t be fresh and you don’t want to use osmium tetroxide, what alternate fixative can be used for lipids?

Answer 28

10% Neutral Buffered Formalin.

Again, do not put section in alcohol fixative or in any solvents (alcohol or xylene) because it will dissolve the lipids and nothing will be stained.

Question 29

How is Oil Red O solution made?

Answer 29

To make up Oil Red O:

1. Oil Red O & Isopropanol (98%) is used to make up the stock solution.
2. The working solution uses 24 mL of stock solution & 16 mL of distilled water.

Question 30

What happens to the dye molecules when a tissue of lipids is put in a solution of Oil Red O?

Answer 30

The Oil Red O dye molecules go and attach to the neutral lipids leaving the alcohol and water left in the solution.

Question 31

What needs to be done after the ammonia water (blueing step)?

Answer 31

After blueing rinse slides in tap water & immediately coverslip slides with an aqueous mounting medium.

Question 32

What trick can you use to seal edges of coverslip?

Answer 32

Fingernail polish careful applied to edge.

But not too much, don’t want to dissolve lipids.

Question 33

What is a lyscochrome?

Answer 33

Oil Red O is a lysochrome. It is more soluble in lipid than its solvent, i.e. preferential solubility.

Question 34

How do you prevent Oil Red O forming a precipitate on the slide?

Answer 34

Need to filter Oil Red O and use within 2 hours of being made to avoid precipitates forming on slide.

Question 35

What are the final colours shown in the Oil Red O technique?

Answer 35

Neutral Fats / Hydrophobic lipids = orange/red.
Phospholipids = pink
Background & nuclei = blue