WEEK 3

Question 1

Why examine tissues and cells

Answer 1

we can study:

1) tissue anatomy and crytoarchitecture

2) the distribution of proteins within the tissue

3) pathological changes associated with disease

Question 2

Histological study of tissues is essential for…

Answer 2

1) clinical diagnostic neuropathology

2) basic and translational neuroscience research

Question 3

Tissue sources

Answer 3

1) Animal models:
ADVANTAGES:
- can study different stages of disease
- can study effects of specific mutations
- can assess therapeutic strategies
LIMITATIONS:
- may not fully recapitulate human disease
- ethical concerns

2) Post-mortem donor tissue, pathology samples, surgical surplus
ADVANTAGES:
- reduce need for animal research
- arguably better for studying human disease
LIMITATIONS:
- ethical concerns
- limited tissue supply
- low availability of early stages of disease

Question 4

2 methods of tissue preservation

Answer 4

1) chemical fixation

2) cryopreservation

Question 5

Chemical fixation

Answer 5

• no fixative is ideal for all applications
• most applications use formaldehyde, as it gives the best balance between morphology and stain quality
• fixatives that are best at preserving cellular ultrastructure, such as glutaraldehyde, create bonds between proteins in the tissue but can interfere with the staining process. conversely, acetic acid or methanol produce better staining results but give inferior cellular or tissue morphology, as they disrupt hydrophobic bonds between proteins causing them to precipitate.

Question 6

2 methods of tissue fixation

Answer 6

1) immersion fixation

2) perfusion fixation

Question 7

Immersion fixation

Answer 7

immersing the tissue in a chemical fixative. this fixative solution will diffuse with the tissue over time.

• more applicable for small already dissected samples. larger samples can be fixed this way but with agitation.

Question 8

Perfusion fixation

Answer 8

we can inject fixative as a fluid directly into the circulatory system. this requires an intact circulatory system, but can deliver the fixative very quickly throughout the organ or entire experimental animal via blood vessels. this method is preferred because it gives superior preservation.

Question 9

Factors affecting quality of fixation

Answer 9

1) changes in pH
- affect the reactivity of the fixative

2) length of incubation in fixative
- determined by the rate of the fixative diffusion

3) specimen size
- where possible, samples are dissected so that they are no longer than 5mm cubes, as this facilitates optimal fixation

4) temperature
- fixation at 4 degrees will retard degenerative changes but will also reduce the penetration rate to the fixative. in contrast, room temp fixation accelerates fixative penetration but also potential degenerative changes.

Question 10

Cryopreservation

Answer 10

preservation of tissue structure and components by freezing them rapidly without fixation.

• we can stop tissue degradation by rapidly cooling the sample with dry ice or liquid nitrogen: SNAP FREEZING
• unlike chemical fixatives, this method does not modify protein structure. however, morphology is poorly preserved as ice crystals are formed during the process and can damage cellular structures.
• this method does not permanently fix tissue, so over time degradation will occur and will progress rapidly if the sample is not stored at -80 degrees.

Question 11

Embedding

Answer 11

chemical fixation will not harden the tissue enough to allow thin sections to be cut. the sample is embedded in solid media that will give extra external support for tissue structure, and sufficient rigidity to enable to cutting of thin sections.

Question 12

Embedding media

Answer 12

• plastic resin: hard media that allows for much thinner slices.
• paraffin wax: most common embedding media in histology labs. the sample is impregnated with liquid paraffin wax which hardens and forms a matrix in and around the sample, preventing tissue distortion. it is very versatile and allows for sections of different thicknesses to be cut.
• agarose/gelatin: softer embedding media suitable for the production of much thicker sections.
• commercial, water-soluble, embedding media such as OCT are viscous at room temp but solidify upon freezing and act as a support medium for the tissue. this allows for the tissue to be handled for a short time without risk of thawing. (for cryopreserved tissue)

Question 13

Using paraffin wax: 4 steps

Answer 13

1) you have to dehydrate the tissue since water is not miscible with paraffin wax. this is done by sequential immersions in increasing concentrations of alcohol: 70%, 90%, 100%

2) once dehydrated, the tissue needs to be cleared of alcohol, since alcohol is not miscible with the wax. we remove it completely using a solvent miscible with both alcohol and paraffin wax: xylene or histo-clear

3) one cleared, we need to replace the xylene or histo-clear with molten wax. this is called infiltration.

4) finally, we embed the tissue in a block of paraffin wax. the tissue is oriented in metal moulds containing fresh molten wax and then allowed to cool.

• upon cooling to 4 degrees, the wax blocks are removed from the metal moulds and are ready for sectioning or storage.

Question 14

2 tissue processor types

Answer 14

1) “dip and dunk” machine

2) enclosed tissue processor

Question 15

Dip and dunk machine

Answer 15

• has 12 stations
• specimens are placed in a basked and loaded onto the processor. after predetermined times in each station, the basket is transferred from graded alcohols, to xylene, to melted paraffin wax, before being removed for embedding in molten wax.

Question 16

Enclosed tissue processor

Answer 16

tissues are loaded into a chamber and the processing reagents are sequentially pumped in and out under vacuum to increase processing efficiency. this is a high-throughput processor.

Question 17

Paraffin wax embedding station

Answer 17

specimens are transferred onto a molten wax tray on the embedding station. tissues are then placed in metal moulds filled with molten wax and oriented before placing on the cold plate to set the wax.

Question 18

Sectioning

Answer 18

the process of cutting thin slices from the sample, required for microscopic examination

Question 19

Benchtop rotary microtome

Answer 19

• most common microtome
• thickness of between 3 and 10 microns
• once sections are cut, they are floated onto water in a bath maintained at about 40 degrees. this softens the wax surrounding the tissue sections, so it expands, allowing the sections to become flat.
• sections may then be separated to be mounted onto slides individually or as ribbons.

Question 20

Vibratome

Answer 20

• microtome used for samples embedded in softer media such as agarose or gelatin
• thickness of between 50 to 500 microns
• the sections are cut by a blade vibrating at a high lateral speed but advancing slowly through the sample. sections are collected and are stained as free-floating sections and then mounted on slides for microscopic examination.

Question 21

Cryostat

Answer 21

used for sectioning snap-frozen tissues

• sections are cut using a refrigerated cabinet maintained at 20 degrees containing a microtome
• thickness between 8 to 50 microns
• cut sections are mounted onto slides and can be immediately fixed if the tissue has been frozen as fresh tissue, or they can be stored for later use: -20 degrees short term or -80 degrees for long term storage
• because the tissue is snap frozen, the vitreous water is hard enough to act as the embedding medium for cutting sections. however, one may use an embedding medium if required (like OCT).
• useful for delicate or small samples.

Question 22

Sliding microtomes

Answer 22

• used to section frozen samples without the need for the more expensive cryostat.
• machine that is fitted to the benchtop, and the tissue is kept frozen by blasting it with CO2 gas or cardice (solid CO2).
• thickness of 15 to 200 microns, stained as free floating sections.

Question 23

Nissl staining

Answer 23

• dye staining achieved by ionic interaction between the dye and the tissue component, labeling neurons or negatively charged components like DNA and RNA.
• nucleus isn’t stained, but the cytoplasm is bc that’s where the RER is found. it also does not stain axons and dendrites.

Question 24

Luxol fast blue

Answer 24

• acidic dye used to visualize CNS myelin sheaths in paraffin wax sections.
• can be used to study the loss of myelin in neurodegenerative diseases such as MS.

Question 25

Metal impregnation: Golgi stain

Answer 25

- used to visualize finer structures, such as dendrites and spines. - developed by Golgi and modified by Ramon y Cajal - formalin-fixed tissue is immersed in potassium chromate then silver nitrate - stains a small random subset of neurons in exquisite detail. this produces little background staining, and neurons are stained in dark black or brown, producing high-contrast defined structures such as spines. this color is produced by silver salt deposits produced by the neuron - used to study neuronal morphology or to quantify the number of dendritic spines that a neuron has

Question 26

Antigens

Answer 26

large macromolecules, usually proteins or molecules with a protein component like a glycoprotein or a lipoprotein

Question 27

Epitope

Answer 27

small section of the antigen, an 8-15 amino acid sequence that is recognized by the antibody. each antigen has many potential epitope sites.

Question 28

Immunodetection techniques: aims

Answer 28

1) distinguish between cell types because a specific cell type will express a specific protein 2) visualize a location of proteins within the cell - important because the location of a protein may be altered by disease

Question 29

Monoclonal antibodies

Answer 29

- preparation contains a single antibody type that recognizes a single epitope on the antigen. - produced by immunizing or injecting a mouse with an antigen, the protein we wish to generate an antibody against. this can be a human protein. B-cells are then extracted from the spleen of the animal. These B-cells are then fused with myeloma cells, which are immortal. By fusing them, we generate a hybridoma cell, which can grow indefinitely and secrete antibodies. - not all hybridoma cells will generate the antibody that we want, but some will, and each of these will generate an antibody that interacts with a specific epitope on the protein/antigen of interest. after isolating those hybridoma cells, we can expand them to a population.

Question 30

Polyclonal antibodies

Answer 30

- produced by injecting an antigen of interest into a rabbit. the animal's B-cells will produce antibodies against the antigen, and these antibodies can be found in the serum of the animal. different B-cells will make antibodies against a different epitope of the protein, so the serum will contain a collection of antibodies that recognize different epitopes of the same protein. - they can cross-react with other proteins, since the more epitopes that are recognized by an antibody mixture, the more opportunity there is that any one particular epitope may be found on an entirely different protein.

Question 31

Immunoglobin structure

Answer 31

- antibodies are Y-shaped, and are composed of 4 polypeptide chains: 2 identical copies of a light chain, and 2 identical copies of a heavy chain. - the arms of the Y are the antigen binding site. - 5 types: IgG, IgA, IgE, IgM, IgD. - most antibody reagents are IgG or IgM

Question 32

Immunohistochemistry: Direct method

Answer 32

- antibodies are applied to the tissue section. the primary antibody, so called because it binds directly to the antigen, is directly linked to a reporter molecule. these reporter molecules enable us to visualize the bound antibody. - suitable only for highly expressed proteins, because if there are very few epitopes available, the reporter signal may be too weak for us to see.

Question 33

Immunohistochemistry: Indirect method

Answer 33

- allows us to significantly amplify the reporter signal strength - the primary antibody binds to the antigen. but in this case, this antibody has no reporter link to it. the section is then incubated with a secondary antibody that binds to the primary antibody and has a reporter link to it. - the secondary antibodies are polyclonal, so will react with epitopes all over the primary antibody. thus, multiple secondary antibodies will bind to the primary antibody, and, all of them carry a reporter - so the signal is amplified.

Question 34

Detection methods: Fluorescence

Answer 34

the antibody is linked to a fluorochrome that emits colored light at a specific wavelength when illuminated with UV light. - used to detect the presence of more than one protein at the same time by using 2 or more antibodies each linked to different fluorochromes that emit light at different wavelengths. - detected by fluorescence microscopes

Question 35

Detection methods: Enzyme

Answer 35

the antibody is linked to an enzyme - horseradish peroxidase (HRP). a substrate (chromogen) is then added to the tissue and the enzyme and substrate interact to generate an insoluble colored product at the site of the antigen-antibody complex. - detected by a light microscope

Question 36

Immunohistochemical experiments: factors to consider

Answer 36

1) incorporation of positive and negative controls 2) antigen retrieval 3) blocking of non-specific binding

Question 37

Incorporation of positive controls

Answer 37

- to assess fidelity of the technique and specificity on the primary antibody ex: if we see anti-BRDU staining in the intestine of an animal we've incorporated the protein in, we'll know that the staining procedure worked even if it wasn't seen in the treated animal's hippocampus

Question 38

Incorporation of negative controls

Answer 38

it is sufficient to omit the primary antibody - so an positivity seen is assumed to be caused by non-specific binding of the visualisation reagents.

Question 39

Antigen unmasking/retrieval

Answer 39

fixation techniques can mask or alter epitopes so that they no longer bind to the primary antibody, meaning we can't detect antigens. this can be reversed in 2 ways: 1) heat-induced epitope retrieval 2) protease-induced epitope retrieval

Question 40

Heat-induced epitope retrieval (HIER)

Answer 40

- performed using microwave ovens, pressure-cookers, steamers, or water baths, but also commercial systems designed for it. - carried out with sections immersed in a buffer solution, which resists change to pH.

Question 41

Protease-induced epitope retrieval

Answer 41

- sections are pre-incubated in enzymes such as protinase K, trypsin, and pepsin. - which enzyme you use is by trial and error, and overdigestion can destroy both the antigen and the tissue section. so this method has inherent difficulties. it also only works for a small proportion of antigens.

Question 42

Blocking non-specific binding

Answer 42

- the antibodies we use can sometimes bind to non-specific components in cells and tissues with low-affinity, resulting in false positive signals. this is most seen in polyclonal antibodies. - to prevent this, excess protein is added so they can compete for and block binding to these components. - serum contains a large amount of proteins and can compete out the antibody from binding to sites other than the target epitope. the serum used is of the animal species that was used to raise a secondary antibody. - bovine serum albumin is used and is very effective