Immunohistochemistry

Question 1

What is Luxor Fast Blue?

Answer 1

Is an acidic dye in solution that is used to visualise central nervous system myelin sheaths in paraffin wax sections. With this technique myelin is stained a deep blue

Question 2

Myelin sheath are formed by?

Answer 2

Oligodendrocytes in the central nervous system. Composed of cholesterol and glycoproteins, myelin serves to insulate the axons of neurons, which significantly increases the conduction velocity of action potential.

Question 3

Why Luxor Fast Blue stain is useful in a study of myelinated nerve?

Answer 3

Because tracts in the central nervous system, and changes that may occur to myelination.

Question 4

How was the study by Lovato? What study by Lovato showed?

Answer 4

• They use Luxol Fast Blue and cresyl violet stains.
• malformation of the hippocampus, shown in the box inset, in mice lacking the tumor suppressor neurofibromatosis two gene, which has a role in brain development. By combining the two stains we can visualise both the neuron of cells bodies and their myelinated axons, visualising the structure of the entire brain.

-The complete loss of myelin in the white matter of the mouse spinal cord after contusion. Contusion occurs due to traumatic injury and often causes inflammation and bleeding from blood vessels near the injury site.

Question 5

Golgi stain is often used to study neuronal morphology. How The method can be used?

Answer 5

Can be used on very thick sections of the brain, up to 500 micrometers, or even whole brains. This means that for some neuron types, we can visualise the entire neuron and most of their processes in the same section. We can reconstruct the neuron and see what affects, for example, a neurodegenerative disease may have on its morphology

Question 6

There other way to use Golgi Stain?

Answer 6

Is also often used to quantify the number of dendritic spines that a neuron has. For example, there is one study by Glantz and Lewis, who use the Golgi method to show a loss of dendritic spines on the prefrontal pyramidal neurons with patients with schizophrenia.

Question 7

Myelin:

Oil red O:

Answer 7

Frozen sections only. The majority of flats are destroyed by Paraffin wax processing the dye, when dissolved in 70 per cent alcohol has a preferential solubility for the fat

Question 8

Myelin:

Solochrome cyanine:

Answer 8

Much simpler to use than LFB method giving a similar myelin positivity

Question 9

Myelin:

Osmium tetroxide:

Answer 9

Fixed tissue are immersed in 2 per cent Osmium for 2 hrs and then processed to P. Wax (or snap-frozen and cut as frozen sections)
Oxidation of lipid unsaturated double bonds causes reduction of OsO4 to metallic osmium
Excellent for studying peripheral myelinated nerved fibres

Question 10

Myelin:

March’s method for degenerating myelin

Answer 10

Osmium turns degenerating myelin black. Potassium chlorate prevents normal myelin (mostly) from reacting with Osmium and will be unstained. More complicated than dye stains.