Other Methods/Electron Microscopy/Enzyme Histo

Question 1

What is enzyme histochemistry typically used for?

Answer 1

Diagnosing disease in skeletal muscle.

Question 2

What is a catalyst?

Answer 2

A catalyst is a substance that changes the rate of the reaction of the chemical without being consumed by the reaction.

Question 3

What are enzymes?

Answer 3

Enzymes are proteins that catalyze chemical reactions occurring in biological systems by temporarily combining with their specific substrate or the compound on which they act. Product is formed and they are released. As illustrated by textbook:
E(enzyme) + S (substrate) –> ES (temp comb.) –> P (product) + E (Enzyme)

Question 4

Is muscle fixed before freezing for enzyme histochemistry?

Answer 4

Muscle is not fixed before freezing for enzyme histochemistry.

Question 5

What is quenching in histochemistry?

Answer 5

Snap Freezing.

Question 6

What effect does quenching (snap freezing) have upon the tissue?

Answer 6

1. Causes almost complete inactivation of all chemical processes in the tissue.
2. Brings the tissue to a solid state. (Tissue constituents diffusion is almost stopped completely).
3. Ice crystals form (freezing of unbound water in tissue).

Question 7

What size of ice crystals (qualitative) is desired in quenching (snap freezing)?

Answer 7

You want the ice crystals to be as small as possible so that the muscle cells can be observed. (Other wise it may be impossible for the pathologist to see.)

Question 8

What factors regarding the coolant are important for ice formation?

Answer 8

Size, shape, and thermal properties.

Question 9

How does one get small ice crystals to form?

Answer 9

The more rapid the freezing, the smaller the ice crystals that are formed.

Question 10

What does a slow rate of freezing cause?

Answer 10

1. Large crystals.
2. Empty holes when examined microscopically.
3. Displacement of tissue.

Question 11

How are muscle biopsies oriented in on the cork/chuck (i.e. embedding media)?

Answer 11

Muscle biopsies are placed in gum tragacanth.

Question 12

What is the preferred steps/method of freezing a muscle biopsy specimen?

Answer 12

1. Quickly immerse specimen in isopentane cooled in liquid nitrogen to -150degC.
2. Check temperature of isopentane with a thermometer to ensure it is at -150C before placing specimen in (TB says for ~15 secs).
3. Next place the specimen in liquid nitrogen until ready to cut. Storing at -70C.

Question 13

What happens when a frozen biopsy specimen is ready to cut in the cyrostat?

Answer 13

It is warmed up to -20C, which is the temperature of the cryostat cabinet.

Question 14

What will happen if the frozen specimen is not allowed to warm up to -20C?

Answer 14

It will be too hard to cut and will produce poor sections.

Question 15

What are the typical thickness of frozen tissue specimens are cut at?

Answer 15

6 to 10 um

Question 16

What is done with frozen tissue specimens after they are cut? What is recommended and why?

Answer 16

1. Cut sections are placed on glass slides which can be stored in the freezer.
2. Recommended to stain ASAP for optimum demonstration of enzyme activity.

Question 17

How are the glass slides of frozen sections for enzyme histo stored and transported?

Answer 17

1. Wrapped in plastic and then in foil and stored at -70C.

2. Transported in a “super cooler” in liquid nitrogen.

Question 18

How much can a light microscope magnify tissue versus an electron microscope? Source of power to view?

Answer 18

Light Microscope: 1000x max magnification.
Electron Microscope: >1,000,000x and still have resolution.
Electron microscopes use an electron beam instead of visible light.

Question 19

How small is a tissue cut for fixation for use in electron microscopy?

Answer 19

Tissue is cut to a max 1 mm^3 so fixatives can penetrate the tissue in a timely fashion.

Question 20

What is the primary fixative used in electron microscopy?

Answer 20

Primary Fixative: Glutaraldehyde.
Paraformaldehyde can also be used.
Secondary Fixative: Osmium tetroxide.

Question 21

What affect does the 2nd-ary fixative osmium tetroxide have on the tissue? Health concern for workers?

Answer 21

1. Osmium Tetroxide makes tissue appear black as it stains the lipid in cell membranes black.
2. Very toxic.

Question 22

What happens following fixation of tissues for electron microscopy?

Answer 22

Following fixation the tiny piece of tissue is dehydrated with increasingly concentrated alcohols.

Question 23

What is done after dehydration for tissue processing for electron microscopy?

Answer 23

Next steps are:

1. Transitional fluid is used called propylene oxide to remove any traces of alcohol.
2. Embedded into an epoxy resin in a rubber mold. Hardens at 60C.

Question 24

How long can the tissue processing last for the electron microscopy?

Answer 24

The process can take days to get a final product.

Question 25

What are the steps for tissue microtomy for electron microscopy after tissue processing?

Answer 25

The general steps are:

1. Hardened resin is removed from mold.
2. Trapezoid piece is cut to get tiny sections.
3. Glass knife is used for “rough” sections. Cut on ultra microtome at 0.5 um. No ribbons formed. Cut directly onto tiny waterbath attached to the glass blade.
4. Look through microscope to see if a good section is obtained.

Question 26

What is the “rough section” also called? And what is it used for?

Answer 26

Rough section is also called the survey section.

It is used to find the desired location of the tissue for further examination.

Question 27

What are the steps for determining what part of the survey section to be further isolated?

Answer 27

The steps are:

1. The 0.5 um sections are put on a slide (w/ toluidine blue stain on it) with a glass rod.
2. Section(s) are viewed under a light microscope after it is heat fixed and the best piece of tissue is further isolated by cutting a smaller trapezoid section from the original piece.

Question 28

What kind of knife is used and how thick of sections are cut for the final section used in electron microscopy?

Answer 28

1. Diamond knife to cut an ultrathin section.

2. Sections cut 60 - 90 nm thick.

Question 29

When cutting on an ultramicrotome how does one tell how things are working?

Answer 29

Use a microscope part of the ultramicrotome to see if sections are being cut and floated into the attached waterbath.

Colour changes with tissue thickness. Only silver sections are thin enough to be picked up (gold too thick!).

Question 30

What can be used to remove compressions in an ultrathin section cut for electron microscopy?

Answer 30

Chloroform vapour can be used to remove compressions.

Question 31

After the ultrathin sections of tissues are cut what are the next steps for electron microscopy?

Answer 31

Ultrathin sections are put onto copper support grids. The grid is moved into the waterbath and the tissue is guided onto it.
The grid is dried on filter paper.

Question 32

What kind of stains are used in electron microscopy?

Answer 32

Heavy metals such as uranium or lead.

Very hazardous chemicals.

Question 33

How is the stain applied for electron microscopy?

Answer 33

Tissues are stained by placing the grid into a drop of chemical.
It is washed afterward.

Question 34

How are the specimens viewed? (Or rather what is done with the grid to view the slides)?

Answer 34

The grid is put into a specimen holder and inserted into the electron microscope for viewing. The vacuum is put on.

Question 35

Describe the theory for how heavy metal stains work on a tissue for electron microscopy?

Answer 35

Where there are heavy metals deposited in the electron dense tissue, the electrons cannot pass through. These show as black on the image. Where it is not electron dense (electron light) there are no heavy metal deposits and the electrons can pass through to the detector.