Immunohistochemistry - Week 5

Question 1

what is the definition of immunochemistry

Answer 1

proteins studied in situ within cell/tissues using labelled antibodies

Question 2

what is the definition of immunocytochemistry and immunohistochemistry

Answer 2

immunocytochemistry:
the study of the proteins within cells (variable or fixed)

immunohistochemistry:
the study of proteins within tissues (fixed or frozed)

Question 3

what is the application of immunochemistry

Answer 3

applications:
- cancer diagnosis
> cell markers for proliferation and apoptosis
- Research
> Location(s) is important to study function

Question 4

what are the 3 different types of label and there use

Answer 4

Labels:
- Fluorescent dyes
> used in immunofluorescence, the dye fluoresces under uV when chemically bound to primary and secondary antibody

• Enzymes
  > enzymes converts soluble substrate to insoluble product in order to localise the target protein. examples, horseradish peroxidase and alkaline phosphatase.
• Colloidal gold
  > used with light and electron microscopy

Question 5

why is the washing steps important

Answer 5

it is important to remove unbound antibodies in order to get accurate results.

Question 6

what is fluorescence

Answer 6

an energy emission phenomenon seen in some molecules

Question 7

how do molecules that are promoted to higher energy levels by EM radiation lose energy

Answer 7

molecules promoted to higher energy levels by EM radiation lose energy by:
- EM radiation emitted by the electrons that are transitioning to lower energy levels
- Heat loss due to bond vibration/rotation

Question 8

what are 2 key facts about molecules with aromatic ring structures

Answer 8

Molecules with aromatic ring structures:
- Low bond vibration/rotation
→ their excess energy is lost as fluorescent light.

Question 9

what is the purpose of fluorescent dyes or probes

Answer 9

they are used so you can visualise biological components

Question 10

what are the different things that can be visualised using fluorescent dyes or probes

Answer 10

• Cell surface receptors.
• Intracellular organelles.
• Nucleic acids.
• Apoptosis markers.

Question 11

what are 3 examples of fluorescent dyes that are used

Answer 11

Examples of different types of dyes:
- FITC (Fluorescein isothiocyanate). Abs = 488 nm;
Ems = 518 nm.
- Alexa Fluor® 488.
- DAPI (4’,6-diamidino-2-phenylindole). Used to stain nucleic acids.

Question 12

why are fluorescent proteins used in microscopy

Answer 12

In living cells, fluorescent proteins are most commonly utilized to track the localization and dynamics of proteins, organelles, and other cellular compartments.

Question 13

what are 2 examples of fluorescent proteins

Answer 13

Examples of fluorescent proteins:
GFP (Green fluorescent protein). Emits green light.
PE (Phycoerythrin). Emits red light.

Question 14

how are samples prepared in immunohistochemistry

Answer 14

Fixation
🡥 🡦
Tissue Sectioning 🡢 Section before staining
sample 🡥
🡦 🡥
Freezing

Question 15

how is the tissue sample prepared in 6 steps from start to finish in immunohistochemistry

Answer 15

Tissue Sampling: Quickly after removal from organism to prevent cellular/tissue degradation.

Fixing: Immobilises , kills & preserves cells/tissue.
> Permeabilises cells to staining reagents.
> Cross-links macromolecules – stabilised & fixed in
position.
> Organic solvents e.g. methanol: acetone.
> Reactive aldehydes e.g. formaldehyde.

Embedding: Soft tissue requires treatment with liquid resin/wax → hardens tissue for sectioning.

Freezing: Dry ice (−80°C) or liquid nitrogen (−196°C).

Sectioning: The sample is cut into 1 – 10 µm thick sections with a microtome before mounting on a microscope slide.

Staining: Primary antibody to the protein of interest + labelled secondary antibody or conjugated antibody. Labels are either colourimetric or fluorescent.

Question 16

why are cells permeabilised

Answer 16

in order for intracellular target antigens to be expressed in cytoplasm and/or nucleus the staining antibodies need to transverse cell and/or nuclear membrane thus for the transgression to occur the cells must be permeabilised.

Question 17

what organic solvents used in fixing also permeabilise cells

Answer 17

examples of organic solvents for fixing that also permeabilise are:
- methanol
- acetone

Question 18

what are detergents used to permeabilise cells

Answer 18

• Triton X-100
• Tween 20

Question 19

what are incubation times strictly monitored when cells are being permeabilised

Answer 19

incubation times are strictly monitored because over-exposure can lead to cell destruction

Question 20

how do reagents create holes when cells are being permeabilised

Answer 20

reagents create holes in membranes by dissolving the phospholipid layer

Question 21

why is blocking used in immunochemistry

Answer 21

blocking is used to prevent non-specific binding of antibodies (except to target antigen) to proteins.

Question 22

how does blocking work in immunochemistry

Answer 22

A blocking buffer is a solution of irrelevant protein, a mixture of proteins, or other compounds that passively adsorb to all remaining binding surfaces of the plate. this reduces background interference and non-specific binding.

Question 23

what is commonly used as blocking agents in immunochemistry

Answer 23

• BSA
• casein
• proprietary proteins

Question 24

what is the main benefit of blocking agents in immunochemistry

Answer 24

blocking agents together with washing steps reduces background staining

Question 25

example of immunohistochemistry, which protein used, sample used, antibodies used and substrate used

Answer 25

PAX8 protein: Transcription factor over-expressed in epithelial ovarian cancer cells.

Sample:
The epithelial layer of human ovarian carcinoma.

Antibodies:
Primary: rabbit anti-human.
Secondary: anti-rabbit HRP-labelled.

Substrate:
Diaminobenzidine (DAB).

Question 26

where is L1-CAM and PAX8 coexpressed

Answer 26

L1-CAM and PAX8 are co-expressed in collecting duct cells of normal renal tissue.

Question 27

why are fluorescent microscopes better than light microscopy or colourimetric staining

Answer 27

Fluorescent microscopes allow the merging of such images which is not possible with colourimetric staining and light microscopy

Question 28

how is the tissue sample prepared in 5 steps from start to finish in immunocytochemistry

Answer 28

The extracellular matrix surrounding cells has been removed.

Cells can be:
Isolated from a block of tissue.
Grown in cell culture (adherent and suspension cells).

Cell culture: Cells are grown under controlled conditions outside of their natural environment.

Fixing: Immobilises, kills and preserves the cells.
Permeabilises cells to staining reagents.
Cross-links macromolecules – stabilised & fixed in position.
Organic solvents e.g. methanol: acetone.
Reactive aldehydes e.g. formaldehyde.

Staining: Primary antibody to the protein of interest + labelled secondary antibody or conjugated antibody. Labels can be either colourimetric or fluorescent.