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Neuroscience IV > 3rd week 3 (staining, immunotechniques) > Flashcards

3rd week 3 (staining, immunotechniques) Flashcards

1
Q

Staining methods (4)

A

dyes

heavy metals

fluorochromes

immunotechniques

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2
Q

dye staining, selective but not specific.

What does this mean?

A

A dye is selective, for example, it sticks to Nissl bodies (same as ‘Nissl substance’) in neurons, but it doesn’t specify between the type of neurons. That is possible when using immunotechniques.

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3
Q

Nissl staining / Cresyl Violet Staining

commonly used to identify:

Based on: …. interaction between dye and the tissue

Discovered by Franz Nissl at the end of xx century

Uses basic …. … to stain RNA blue.

A

neuronal structure in brain and spinal cord tissue.

ionic

19th

The Cresyl Violet method uses basic aniline dye to stain RNA blue.

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4
Q

Nissl bodies are rich with … molecules

A

RNA

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5
Q

Anionic stain, cationic stain, (neutral stain)

Explain differences

A

Anionic, or acidic stain

  • Chromogen of acidic stain is negatively charged.
  • are used to stain the positively charged components such as background staining.

histone protein is positively charged so it can be stained by acidic stain.

-can’t stain bacterial cell due to repulsion of same charge.

Examples: Eosin, Nigrosin, India ink

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6
Q

Acidic stain

  • charge
  • uses
  • can’t be used for…
  • exmples (names of stains)

[so, there are positively and negatively charged and neutral stains]

A

Anionic stain

  • Chromogen of acidic stain is negatively charged. so, it is also known as Anionic stain (positive called basic or cationic)
  • are used to stain the positively charged components such as background staining.
  • histone protein is positively charged so it can be stained by an acidic stain.
  • can’t stain bacterial cell due to repulsion of the same charge.

Examples: Luxol, fast blue, Eosin, Nigrosin, India ink

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7
Q

Cationic stain

  • charge
  • uses (combines with)
  • examples (names of stains)
A

Basic stain (Cationic stain)

Chromogen or coloured part of basic stain is positively charged. so, it is also known as cationic stain.

  • Basic stain are used to stain negatively charged components such as RNA, DNA, bacterial cell.
  • crystal violet, cresyl (methylene blue, safranin, malachite green,basic fuschin)
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8
Q

Cresyl violet

Cresyl violet stains the … components of the neuronal cytoplasm a violet colour, specifically … bodies. Often used in brain research.

Are DNA and RNA anions or cations?

A

Cresyl violet[edit]

Cresyl violet stains the acidic components of the neuronal cytoplasm a violet colour, specifically nissl bodies. Often used in brain research.

RNA and DNA are acidic - anions (stained

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9
Q

Why does basic stain (like cresyl violet) stain Nissl bodies dark purple and nucleus lighter?

A

Basic/cationic stains bind to negative molecules. RNA and DNA are negative, nucleus less negative - lighter colour.

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10
Q

Why is Nissle staining used for neurons and not so much for other cell types?

Give examples of type of studies? (2)

A

Neurons rich with DNA, RNA, other cells have less.

Neural loss

Abnormal growth

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11
Q

Luxol fast blue

  • basic (cationic) or acidic (anionic) stain?
  • mechanism?
  • example, often used for what part of a neuron?
  • what conditions?

Explain two colours in the picture

A
  • Acidic (anionic)
  • dye (anion) binds to positively charged molecules (cation)
  • to stain myelin lipoproteins (oligodendrocytes, cholesterol, glycoprotein)
  • neurodegeneration, myelin sheat loss like MS
  • combining Nissl staining and Luxol blue shows bot bodies and axons
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12
Q
A
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13
Q

Metal impregnation

Golgi stain, used for what part of the neuron?

Camillo Golgi in 17xx, what role has Santiago Ramon y Cayal

…. fixed tissue is immersed in … chromate, then silver …..

  • color produced by …. salt deposit in the neuron
  • this technique causes little/lot background staining
  • used often in neuronal …., thick sections up to xxx microns, even the whole brain.
A
  • dendritic spines
  • Golgi 1783, Cayal modified later
  • formalin-fixed tissue is immersed in potassium chromate, then silver nitrate
  • silver salt
  • little
  • neuronal morphology, up to 500 microns
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14
Q

These methods are often used for which part of a neuron:

Oil red O:

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

Solochrome cyanine:

• much simpler to use than LFB method giving a similar myelin positivity

Osmium tetroxide:

  • fixed tissues are immersed in 2 per cent Osmium for 2hrs 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 nerve fibres

Marchi’s method for degenerating myelin

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

A

Myelin

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15
Q

Silver methods used for which type of cells?

Several methods utilising Silver nitrate which precipitates as metallic silver on axons: Holmes, Bielschowsky –type methods.

A

Neurons

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16
Q

What type of cells?

Cajal’s gold-sublimate method for astrocytes, Holzer’s and Mallory’s PTAH methods for gliosis (astrocyte fibrosis/scarring)

For many of the old staining methods replaced by:

A

Gliall

immunomethods utilising antibodies that have been raised against epitopes specific for a comprehensive range of neural proteins including Neurofilaments (neurons), Glial fibrillary acidic protein (astrocytes, ependymal cells), Iba1 (microglia), Myelin basic protein (myelin) and OLIG2 (oligodendrocytes).

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17
Q

Immunotechniques

targets ….. visualised by ….

(remember:

….. induces an immune response

That leads to production of …..)

A

targets antigens, visualised by antibodies

—

(remember:

antigens induce an immune response

leads to the production of antibodies)

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18
Q

Two types of detection methods in immunotechniques

A

enzyme based

fluorescent based

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19
Q

Epitope?

A

sequence (part) of antigen, recognised by antibody

(8-15 amino acids)

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20
Q

Advantage of immunodetection?

A

Makes it possible to distinguish between specific cell types and location (vrt. dye staining selective but not specific)

21
Q

Two types of antibodies

….clonal

…. clonal

A

monoclonal

polyclonal

22
Q

monoclonal antibody

A

Single antibody

recognises a single antibody on the antigen

23
Q

Producing monoclonal antibodies

mouse is injected with….

In the spleen of the mouse …. produce ….

These are extracted from the mouse spleen and …. with for example myeloma cells (human cancer cells)

[why]

  • the cell in this stage is called …. What does it do?
  • what do the selected ones produce?

used in?

A
  • antigen (this can be a human protein)
  • B cells produce antibodies
  • fused

[B cells can’t divide, but cancer cells can divide rapidly. Needed in the next stage to produce a large number of antibodies]

  • combination of mouse B cell and a cancer cell is called a HYBRIDOMA
  • secrets antibodies, the ones needed are selected, and antobody producing hybridomas are cloned
  • monoclonal antibodies

research, cancer treatment pregnancy test etc.

24
Q
A
25
Q

Disadvantage of monoclonal antibodies in therapy?

A

Too specific. [Antigen have many proteins]

26
Q

Polyclonal antibodies

A

A antigen protein has many epitopes. B cells make many antibodies.

Serum of an animal polyclonal.

27
Q

What are:

igG, igM, igA, ind, igE

most common?

A

immunoglobulins, antibody types

most common igG

28
Q

Antibodies have a binding site for…

A

antigen epitopes

on a antigen cell surface there’s a protein, an epitope that an antibody can bind to.

[Antigen has a binding site that matches the antigen epitope on it’s surface]

29
Q

DIRECT way of antibodies binding?

What is suitable for direct binding

INDIRECT binding

Suitable for what, effect?

A

Antibody binds to epitope on an antigen.

Antibody has a reporter molecule

Suitable for highly expressed proteins [when antibody binds directly to antibodies with high quantities theres enough reporter molecules showing where the badies are]

INDIRECT

polyclonal/secondary antibodies bind to primary antibodies. More reporter molecules from one antigen, amplifies the reporter molecule signalling.

30
Q

antibody reporter molecule can be a (2)

A

enzyme or fluorochrome

31
Q

Fluorescence method

fluorochrome - reporter molecule

What is the mechanism behind the colour?

A

Fluorochrome is not coloured but it emits coloured light at certain wavelength of UV light.

CAn be seen with fluorescence microscope

32
Q

How can the same picture have different fluorescent colours?

A

When two or more antibodies are used, different fluorochromes emit different colours (different UV wavelength for each colour)

This makes detecting more than one protein possible

33
Q

Fluorescence method is used together with which staining method?

A

Immunotechniques, antibodies - reporter molecule is the one with a fluorochrome

34
Q

Enzymatic detection

How does it work?

A

Antibody reporter molecule can be an enzyme. Often horseradis perioxidase is used as the enzyme linking to an antibody.

This acts with a chromogen added to the tissue. The combination of the enzyme and chromogen can be seen with a microscope light.

35
Q

Immunostaining

What needs to taken into account (planned)

(3)

A

1) Incorporation of positive and negative controls

2) Antigen retrieval
3) Blocking of non-specific binding

36
Q

Adding positive controls

What is BrdU?
What is it used for?

A

synthetic analogue of a nucleotide thymidine

(on of the 4 nucleotides used to produce DNA)

Can be used to create a positive control in immuno staining

37
Q

BrdU, positive control

Explain the procedure

Where is this often used?

A

-BrdU injected to mouse

BrdU used in DNA synthesis

Can be detected by using anti-BrdU antibody

  • adult stem cell neurogenesis in hippocampus
38
Q

What is the use of positive control?

A typical place to check for positive control results

A

If there’s no ‘blobs’ in staining, what went wrong?

Did the staining not work or are there nothing to find?

Adding a positive control shows that the staining has worked, the control is showing a result e.g staining did work.

  • in the small intestine where there is always cell division happening, nucleotides are being used, also the synthetic and marked one. Will stain.
39
Q

What causes negative control results?

A

Unspecific binding

Getting results when the primary antibody is left out and there should not be any results.

40
Q

What problem can tissue fixation cause for using immune techniques for staining?

A

Fixation procedures can mask or alter epitopes so that they can no longer bind to the primary antibody.

41
Q

What is antigen unmasking or retrieval?

What causes ‘masking’?

A

Antigen unmasking or retrieval refers to any technique where the masking of an epitope is reversed so that the antibody can again bind to it.

Tissue fixation

42
Q

Antigen unmasking (retrieval) methods (2)

A

Heat-induced epitope retrieval (HIER)

(microwave, pressure cooker,water bath etc. why HIER works is unknown). Usually done section immersed in buffer solution. Keeps pH. Buffer solutions:

Citric acid pH6

Citrate buffer pH6

Tris pH9

Tris/EDTA pH9

EDTA pH8

Tris pH10

Protease-induced epitope retrieval

sections are pre- incubated in enzymes, right one found with trial and error.

Proteinase K

Trypsin

Pepsin

43
Q

non specific binding

More often happens in monoclonal antibodies / polyclonal antibodies

What can be used to block non-specific binding? (2)

A

polyclonal antibodies [will bind to a protein that it was supposed to bind to, gives a false result (like there’s more of it, some is false binding]

Serum from the animal that was used to get the primary antibodies from. Serum contains proteins that bind to the non-specific sites.

Excess protein like BSA (bovine serum albumin):

competes with antibodies for non- specific binding sites

44
Q

Explain the techniques used to get 3 colours (picture)

(Paraffin wax section from a mouse model of Alzheimers Disease showing astrocytes surrounding an amyloid plaque.)

A
  • double indirect immunofluorescence staining
  • incubated simultaneously with two primary antibodies that each recognised a different

protein (glial fibrillary acidic protein (GFAP) and beta amyloid) ( makes amyloid plaques, cause of Alzheimers in red)

  • Nuclei are stained with a fluorescent blue stain called DAPI.
45
Q

To consider when performing immunostaining? (3)

A

1) Inclusion of positive and negative controls

2) Do I need to use antigen retrieval?
3) Blocking of non-specific binding

46
Q

What is stained with an anti-GFAP antibody?

A

glial fibrillary acidic protein (GFAP)

Astrocytes

47
Q

Possible with immunohistochemistry:

analyse ….. distribution in the cells

determine whether pathological ….. have occurred and we can analyse …… features

A

analyse protein distribution in the cells, we can determine whether pathological changes have occurred and we can analyse morphological features

48
Q
A

Neuroscience IV (9 decks)

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