3rd week 3 (staining, immunotechniques) Flashcards
Staining methods (4)
dyes
heavy metals
fluorochromes
immunotechniques
dye staining, selective but not specific.
What does this mean?
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.
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.
neuronal structure in brain and spinal cord tissue.
ionic
19th
The Cresyl Violet method uses basic aniline dye to stain RNA blue.
Nissl bodies are rich with … molecules
RNA
Anionic stain, cationic stain, (neutral stain)
Explain differences
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
Acidic stain
- charge
- uses
- can’t be used for…
- exmples (names of stains)
[so, there are positively and negatively charged and neutral stains]
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
Cationic stain
- charge
- uses (combines with)
- examples (names of stains)
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)
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?
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
Why does basic stain (like cresyl violet) stain Nissl bodies dark purple and nucleus lighter?
Basic/cationic stains bind to negative molecules. RNA and DNA are negative, nucleus less negative - lighter colour.
Why is Nissle staining used for neurons and not so much for other cell types?
Give examples of type of studies? (2)
Neurons rich with DNA, RNA, other cells have less.
Neural loss
Abnormal growth
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
- 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
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.
- 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
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
Myelin
Silver methods used for which type of cells?
Several methods utilising Silver nitrate which precipitates as metallic silver on axons: Holmes, Bielschowsky –type methods.
Neurons
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:
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).
Immunotechniques
targets ….. visualised by ….
(remember:
….. induces an immune response
That leads to production of …..)
targets antigens, visualised by antibodies
—
(remember:
antigens induce an immune response
leads to the production of antibodies)
Two types of detection methods in immunotechniques
enzyme based
fluorescent based
Epitope?
sequence (part) of antigen, recognised by antibody
(8-15 amino acids)
Advantage of immunodetection?
Makes it possible to distinguish between specific cell types and location (vrt. dye staining selective but not specific)
Two types of antibodies
….clonal
…. clonal
monoclonal
polyclonal
monoclonal antibody
Single antibody
recognises a single antibody on the antigen
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?
- 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.
Disadvantage of monoclonal antibodies in therapy?
Too specific. [Antigen have many proteins]
Polyclonal antibodies
A antigen protein has many epitopes. B cells make many antibodies.
Serum of an animal polyclonal.
What are:
igG, igM, igA, ind, igE
most common?
immunoglobulins, antibody types
most common igG
Antibodies have a binding site for…
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]
DIRECT way of antibodies binding?
What is suitable for direct binding
INDIRECT binding
Suitable for what, effect?
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.
antibody reporter molecule can be a (2)
enzyme or fluorochrome
Fluorescence method
fluorochrome - reporter molecule
What is the mechanism behind the colour?
Fluorochrome is not coloured but it emits coloured light at certain wavelength of UV light.
CAn be seen with fluorescence microscope
How can the same picture have different fluorescent colours?
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
Fluorescence method is used together with which staining method?
Immunotechniques, antibodies - reporter molecule is the one with a fluorochrome
Enzymatic detection
How does it work?
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.
Immunostaining
What needs to taken into account (planned)
(3)
1) Incorporation of positive and negative controls
2) Antigen retrieval
3) Blocking of non-specific binding
Adding positive controls
What is BrdU?
What is it used for?
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
BrdU, positive control
Explain the procedure
Where is this often used?
-BrdU injected to mouse
BrdU used in DNA synthesis
Can be detected by using anti-BrdU antibody
- adult stem cell neurogenesis in hippocampus
What is the use of positive control?
A typical place to check for positive control results
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.
What causes negative control results?
Unspecific binding
Getting results when the primary antibody is left out and there should not be any results.
What problem can tissue fixation cause for using immune techniques for staining?
Fixation procedures can mask or alter epitopes so that they can no longer bind to the primary antibody.
What is antigen unmasking or retrieval?
What causes ‘masking’?
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
Antigen unmasking (retrieval) methods (2)
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
non specific binding
More often happens in monoclonal antibodies / polyclonal antibodies
What can be used to block non-specific binding? (2)
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
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.)
- 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.
To consider when performing immunostaining? (3)
1) Inclusion of positive and negative controls
2) Do I need to use antigen retrieval?
3) Blocking of non-specific binding
What is stained with an anti-GFAP antibody?
glial fibrillary acidic protein (GFAP)
Astrocytes
Possible with immunohistochemistry:
analyse ….. distribution in the cells
determine whether pathological ….. have occurred and we can analyse …… features
analyse protein distribution in the cells, we can determine whether pathological changes have occurred and we can analyse morphological features
