Animal Ethics & Immunostaining techniques + imaging

Question 1

What is animal ethics?

Answer 1

The moral principles that govern how we treat animals that are experimented on.

Question 2

What is morality?

Answer 2

is considered right and acceptable treatment of animals in research

Question 3

What are the 5 main moral theories?

Answer 3

5 main moral theories:
Contractarianism
Utilitarianism
The relationship view
The animal rights point of view
The respect nature point of view

Question 4

What is Contractarianism?

Answer 4

A type of moral theory that employs the idea of contracts between individuals to govern their interactions.

Question 5

What is Utilitarianism : Cost V Benefit?

Answer 5

Actions are right if they are useful or beneficial to the majority

Question 6

What is ‘The Relational View’?

Answer 6

• Highlights the importance of our relationships to animals and is based upon considering animals in a sort of hierarchical order.
• Have special duties to domestic animals because they are in our care
• Considers how our treatment of animals might affect our treatment of humans.

Question 7

What is ‘The Animal Rights View’?

Answer 7

• Directly opposes the concept of animal use, puts clear and definitive limits on our treatment of animals (abolitionist position)
• Animals should have the same considerations as human beings, the right not to suffer or be killed for human benefit.

Question 8

What is ‘The Respect for Nature View’ ?

Answer 8

The moral value of species. We have a duty to protect not just individual animals, but the species to which they belong – and, in particular, the integrity of each species.The preservation of species is morally good, should respect nature and its rich genetic structures. Not genetically modify species (disrespectful interference), selective breeding.

Question 9

How do ‘in-vitro experiments’ work?

Answer 9

Many different forms
Cheaper
Quicker
iPSCs/organoids very promising
Interesting work in wound repair for MIs
Can build a scaffold of myocardial cells
Cannot fully replicate a living system

Question 10

What are the disadvantages and advantages in computer modelling?

Answer 10

-can be an alternative to animal experiments
-Basic understanding needed before programming possible
-Limited processing power
-Can’t program a computer to mimic physiology until we have seen the real thing in action.

Question 11

Why use animals in research?

Answer 11

-Systems similar to humans
-Side effects and efficacy of drugs only show up in in vivo tests
-Whole system complexity of interactions only replicable in vivo
-Law says so!

Question 12

What animals are used in research?

Answer 12

horses, monkey, mice, zebrafish, chicks, rats

Question 13

What is the Animals Scientific Procedure Act (ASPA) 1986?

Answer 13

-Act of UK Parliament that permits the use of animals in scientific procedures
-Regulates the use of protected animals in any experimental or other scientific procedure which may cause pain, suffering, distress or lasting harm to the animal.

Question 14

What is the 3 tier licensing system authorised by Home office?

Answer 14

• Establishment licence – certificate of designation
• Project licence – specific research/testing programme
• Personal licence – specific individual/competency

Question 15

When are the licences for animal work approved?

Answer 15

-Benefit outweighs cost.
-If there is no non-animal alternative.
-Minimum number of possible animals used.
-Using animals with lowest sensitivity to pain possible.
-Pain is minimised.
-Research premises have necessary facilities to care for animals.

Question 16

Who approves animal work?

Answer 16

LEC

Question 17

What are the 3 R’s?

Answer 17

-Replacement
Alternative techniques

-Reduction
Minimum number
Fewer animals
More information

-Refinement
Better housing
Improve procedures
Welfare

Question 18

What is immunohistochemistry?

Answer 18

technique used to identify specific proteins or other molecules in tissues by using antibodies.

Question 19

What is immunocytochemistry?

Answer 19

similar to immunohistochemistry, but instead of using tissue samples, it involves studying individual cells in culture or in suspension.

Question 20

What are primary and secondary antibodies?

Answer 20

A primary antibody is an antibody that directly binds to the antigen (protein or other molecule) of interest. A secondary antibody is an antibody that binds to the primary antibody. Secondary antibodies are used to amplify the signal produced by the primary antibody, making it easier to detect and quantify the target antigen. it is normally tagged with a marker.

Question 21

What are polyclonal and monoclonal antibodies?

Answer 21

Monoclonal antibodies are antibodies that are produced by a single clone of B-cells and are therefore highly specific for a single antigenic epitope. Polyclonal antibodies are antibodies that are produced by multiple clones of B-cells and recognize multiple epitopes on the antigen. They can detect detect multiple antigens, but they may also have lower specificity and affinity compared to monoclonal antibodies.

Question 22

How are secondary antibodies used in fluoresence microscopy?

Answer 22

To detect the presence of a specific protein or other molecule in a sample. In this technique, the primary antibody is first added to the sample, where it binds to the target molecule. The secondary antibody is then added, and it binds to the primary antibody.

The secondary antibody is conjugated with a fluorescent dye, such as fluorescein isothiocyanate (FITC), rhodamine, or Alexa Fluor, which emits light when excited by a specific wavelength of light. When the secondary antibody binds to the primary antibody, it brings the fluorescent tag in close proximity to the protein of interest, resulting in fluorescence at the location of the target protein.

Question 23

What is widefield fluorescence microscopy?

Answer 23

uses a widefield illumination source, such as a mercury arc lamp or a high-powered LED, to excite fluorescent molecules in a sample. In this technique, the entire field of view is illuminated with light, and the emitted fluorescence is detected using a camera or other imaging device.

Question 24

What are the basic components of a widefield fluorescence microscope?

Answer 24

• the illumination source
• a filter set that allows the excitation light to pass through while blocking the emission light
• an objective lens that focuses the excitation light onto the sample and collects the emitted fluorescence
• camera or other imaging device to capture the fluorescence signal.

Question 25

What is confocal microscopy?

Answer 25

uses a laser beam to excite fluorescent molecules in a sample and a pinhole aperture to selectively detect light emitted from a specific plane within the sample. The technique creates high-resolution images of thin optical sections within a sample and can be used to generate 3D reconstructions of biological structures.

In confocal microscopy, the sample is illuminated with a laser beam, which is focused onto a single point within the sample by a lens. The fluorescent molecules in the sample are excited by the laser beam and emit light, which is collected by a detector, such as a photomultiplier tube or a camera, after passing through a pinhole aperture. The pinhole aperture allows only light emitted from a specific plane within the sample to reach the detector, resulting in high-resolution images of thin optical sections.

Question 26

What are the advantages of confocal microscopy over widefield?

Answer 26

improved resolution, increased signal-to-noise ratio, and the ability to generate 3D reconstructions of biological structures.

Question 27

What is selective plane illumination microscopy (SPIM)/Light sheet microscopy?

Answer 27

imaging technique that uses a thin sheet of light to illuminate a sample and a separate objective lens to capture the emitted fluorescence. This technique generates high-resolution 3D images of biological samples with minimal phototoxicity and photobleaching, making it useful for imaging live cells and tissues. In SPIM, the sample is immersed in a transparent medium, such as water or agarose gel, and is illuminated by a thin sheet of light from the side using an objective lens. The emitted fluorescence is collected by a separate objective lens that is positioned at a right angle to the illumination lens.

Question 28

How can dyes be used to fluorescently label specific sub-cellular structures?

Answer 28

1. use a dye called Dapi to label DNA. Dapi intercalates between the DNA bases, and makes the DNA glow when exposed to a UV beam of light. A similar dye, called Sytox-orange, can be used, and this one makes the DNA glow red.
2. Another structure that we often label with dyes is the cortical cytoskeleton. The cortical cytoskeleton is located, as its name indicates, in the cell cortex, underneath the cell membrane. The main components of the cortical cytoskeleton are filamentous actin and myosin, and by labelling these molecules we can highlight the contour of the cells. Phalloidin is a compound that binds to filamentous actin. Forms of phalloidin tagged to various different fluorophores are commercially available. Filamentous actin is not only found in the cortical cytoskeleton, but also in other specialised structures, such as muscle filaments, and phalloidin can be used to highlight those structures as well.

Question 29

What is flow cytometry?

Answer 29

technique used in biology and medicine to analyze and sort cells based on their physical and chemical properties.

Question 30

How does flow cytometry work?

Answer 30

1. Sample preparation: The sample to be analyzed is typically a suspension of cells that has been prepared from a biological sample, such as blood or tissue. The cells are usually stained with fluorescent dyes or antibodies that bind to specific proteins on the cell surface or inside the cell, allowing them to be detected by the flow cytometer.
2. Flow cell: The flow cell is a narrow channel through which the cell suspension is passed in a stream of fluid. As the cells flow through the channel, they are illuminated by one or more laser beams.
3. Optical detectors: The flow cytometer contains several optical detectors that measure different properties of the cells, such as their size, granularity, and fluorescence. As the cells pass through the laser beams, they scatter light in different directions, and the detectors measure the intensity of the scattered light.
4. Data analysis: The flow cytometer generates large amounts of data about the properties of each cell in the sample. This data is typically analyzed using specialized software that can identify and quantify different cell populations based on their unique characteristics.

Question 31

What are fluorophores?

Answer 31

chemical compound that accepts a photon and emit a photon as a result. can allow structures too small to be seen by transmitted light

Question 32

What is Stokes Shift?

Answer 32

Stokes shift is a phenomenon that occurs when a molecule absorbs light at a certain wavelength and then emits light at a longer wavelength.

Question 33

What is forward scatter in Flow cytometry?

Answer 33

One of the measurements obtained during flow cytometry. It refers to the intensity of light that is scattered in the forward direction as a cell passes through the laser beam in a flow cytometer.

As a cell passes through the laser beam, the light is scattered in all directions, but most of it is scattered in the forward direction. The FSC detector in the flow cytometer measures the intensity of this forward-scattered light, which is proportional to the size of the cell.

Larger cells scatter more light in the forward direction than smaller cells, so the FSC measurement can be used to estimate the size of cells in a sample.

Question 34

What is side scatter in Flow cytometry?

Answer 34

Refers to the intensity of light that is scattered at right angles to the laser beam as a cell passes through the flow cell of a flow cytometer.

As a cell passes through the laser beam, some of the light is absorbed or scattered in all directions, including to the sides of the cell. The SSC detector in the flow cytometer measures the intensity of this side-scattered light, which is proportional to the granularity or complexity of the cell.
The more complex internal structure, such as those containing organelles or granules, scatter more light in the side direction than cells with a simpler structure.