Histo Methods

Question 0

What type of sample is curettage biopsy used for?

Answer 0

scrapping from uterus

Question 1

what are the seven steps involved in preparation of a histological sample.

Answer 1

1. tissue collection 2. fixation 3. dehydration and clearing 4. embedding 5. sectioning 6. mounting and staining 7. viewing

Question 2

what is the goal of fixation?

Answer 2

to preserve tissue sample and prevent degradation by either proteolytic enzymes or microorganisms such as bacteria, mold, etc.

Question 3

Why is dehydration generally no longer used as a main method of fixation?

Answer 3

It does not preserve the fine details.

Question 4

How does formaldehyde preserve tissue and what technique is it used with?

Answer 4

Aldehydes have reactive groups that cross-link 3 amino acid groups on proteins, and nitrogenous groups. Basically cross-link every single molecule, one to another within the tissue. Once cross-linked becomes inactivated and loses biological activity. Prevents degradation by endogenous enzymes and creates unfriendly environment for bacterial enzymes. Used with light microscopy.

Question 5

What are the benefits of using glutaraldehyde for fixation? What technique is it used with?

Answer 5

Stronger fixative than formaldehyde. Can preserve sub-cellular structures. Used with electron microscopy.

Question 6

When is dehydration used for fixation? What are the steps?

Answer 6

Used only when you have monolayer, such as cell culture or when you have good access to tissue. Add 80% concentrated alcohol to tissue and leave for short time period. Removes water while proteins precipitate.

Question 7

How does rapid freezing work? What are the benefits?

Answer 7

Use liquid isopentane at -160 degrees C or liquid nitrogen. Brings all biological activity to a halt. If you place in a freezer in -80 degrees can keep for long periods of time. Can be brought back to room temp. and biological activity returns- good for histochemical and immunological stains.

Question 8

What are the disadvantages of using acrylic resin for mounting? What technique is it primarily used for?

Answer 8

Much harder material. Really hard to get rid of. Not compatible with aqueous stains-can’t easily access material. Generally used with electron microscopy because can cut very thin sections (60-80 nm). Takes several days to a week.

Question 9

What are the advantages of fixing tissues as frozen sections?

Answer 9

When sample is heated to room temp. enzyme activity returns and can be used for histochemical and immunological stains. Very rapid (30min-1 hour). Used during tumor excision surgery. Disadvantage: low resolution; 12 microns to 20.

Question 10

What size sections can you cut with paraffin wax mounting and how long does it take?

Answer 10

5 microns-8 microns. 24 hours.

Question 11

If you stain a tissue sample with hematoxylin and eosin (H&E) what structures would you see as pink and which as blue?

Answer 11

H: stains acidic structures BLUE: RNA, DNA, ribosomes, cartilage matrix
E: stains basic structures PINK: proteins, cytoplasm, collagen fibers

Question 12

What is Wright’s stain used for? How is it different from H&E?

Answer 12

Hematologists use it to differentiate blood cells. Use methylene blue instead of hematoxylin.

Pink: erythrocytes, eosinophil granules
Blue: nuclei of WBC, cytoplasm of monocytes and lymphocytes

Question 13

What color will iron hematoxylin stain tissue that has striations of muscle, nuclei and erythrocytes?

Answer 13

Black

Question 14

If I have a sample and would like to identify glycogen stores, what stain should I use?

Answer 14

Periodic Acid Schiff (PAS): will color carbohydrate rich molecules pink.

Question 15

If I have an H&E stained tissue, what color would you expect the nucleus and why?

Answer 15

Blue. Because DNA is acidic.

Question 16

What is the total magnification of a light microscope with objective lens 20x and ocular lens magnification at 10x?

Answer 16

200 x

Question 17

What is resolution?

Answer 17

ability of an instrument to distinguish two points as they come closer and closer together.

Question 18

What are the typical limits of resolution for a light microscope?

Answer 18

.25 microns

Question 19

If I am using a light microscope will I be able to resolve RBC? polio virus?

Answer 19

You can see the RBC (7.5 microns) but not the polio virus which is 28 nm.

Question 20

What is the limit of resolution for TEM?

Answer 20

.2nm

Question 21

What is the resolution limit for SEM?

Answer 21

10 nm

Question 22

What marker can you use to identify the size of objects within a tissue sample?

Answer 22

RBC, which are 7.5 microns (um).

Question 23

Why do erythrocytes (RBC) show up as pink on an H&E stain?

Answer 23

Because they have no nucleus (blue). Hemoglobin is a protein that shows up as pink.

Question 24

Why does cytoplasm show up as ragged, slightly blue/pink in H&E stain?

Answer 24

Because macrophage are undertaking immune surveillance (with acidic vesicles breaking down molecules) this causes a blue reaction. Typically cytoplasm is pinkish.

Question 25

In a Wright Stain what structure shows up as orange-y? What about pink with distinct granules?

Answer 25

Erythrocytes do not have a nucleus and show up as blobs of orange. Basophils (basophilic) have granules and nucleus and are clearly visible. Neutrophils do not have much affinity for either eosin or methylene blue.

Question 26

What is the role of a goblet cell? Why would you see it on PAS stain?

Answer 26

Goblet cells secrete mucus. Made up of large chains of carbohydrates.

Question 27

Why do microvilli show up as pink in PAS?

Answer 27

Have surface coat of carbohydrates (glycocalyx)

Question 28

If you wanted to look at metabolic activity in a cell, what visualization technique would you use?

Answer 28

Autoradiography. Radio labeled precursor. Example: mouse salivary gland; black silver grains reveal where cells have taken up fucose (sugar) and incorporated into glycoproteins.

Question 29

What visualization technique is most appropriate for identifying the location of a specific enzyme?

Answer 29

Histochemistry. Can localize an enzyme by binding the enzyme with a substrate, that when cleaved by the enzyme produces an insoluble colored rxn product (precipitate) that gets deposited at the location of the enzyme.

Question 30

What is the best technique to visualize macromolecules?

Answer 30

Immunocytochemistry. Binding of labeled antibodies. Can be either Direct (antibody itself is florescence labeled) or Indirect (secondary antibody that binds to primary antibody complex).

Question 31

What are the benefits of transmission electron microscopy. How is it different from light microscopy?

Answer 31

Instead of using light, use short-wave electrons. Visualize cellular and sub-cellular structures. Dyes used with light microscopy don’t work; need to impregnate sample with heavy metals (lead, uranium).

Question 32

If you wanted to see surface images of a sample, what visualization technique should you use?

Answer 32

Scanning electron microscopy (SEM).