Immunocytochemistry

Question 1

What is immunocyto(histo)chemistry?

Answer 1

Identification of tissue constituent (antigen) in situ by means of a specific antigen-antibody reaction tagged by a visible label (e.g., fluorophore)

Question 2

What does in situ mean?

Answer 2

Directly at site. Intact cells and tissue.

Question 3

What are the advantages of immunocytochemistry?

Answer 3

Can visualise exact location of antigen which is not possible with other biochemical techniques. Visualise antigens in low concentrations or in limited samples. Not detectable through western blots for example.

Question 4

What are the disadvantages or immunocytochemistry?

Answer 4

Doesn’t show if protein is active or not. E.g., when visualising an enzyme. Doesn’t clarify whether the antigen is being produced at site of the location (need mRNA technology).

Question 5

What do we need for immunostaining?

Answer 5

Antigen (substance capable of immune response). Epitope/antigenic determinant, antibody.

Question 6

What is an epitope?

Answer 6

Part of an antigen that combines with antigen binding site of antibody. Proteins that differ by a single ammino acid or 2 optical isomers can still be distinguished.

Question 6

What is the basic structure of antibodies?

Answer 6

4 polypeptide chains. 2 light chains 220 aa. 2 heavy chains 440 aa.

Question 7

What is the tail or FC of heavy chain for?

Answer 7

Flexible hinge regions to allow the distance between binding sites to be varied. Increases binding efficiency.

Question 7

What determines an antigen being multivalent?

Answer 7

If two or more determinants. Polymer with repeat structures.

Question 8

What is affinity?

Answer 8

Strength of binding to a single antigenic determinant. Independent of number of binding sites.

Question 8

What is avidity?

Answer 8

Total binding strength of all binding sites together.

Question 9

What does equilibrium point depend on?

Answer 9

Concentrations of Ab and antigen strength of interaction.

Question 10

Why split antibody molecules with proteolytic enzymes?

Answer 10

Enhance performance of certain procedures.

Question 11

What are 3 advantages of antibody fragments?

Answer 11

Reduced nonspecific binding from Fc interactions (many cells have receptors that bind Fc region) Ability to control binding in experiments with immunoprecipitation or western blot. More efficient penetration of tissue sections.

Question 12

What are 4 more advantages of antibody fragments?

Answer 12

Higher sensitivity of antigen detection in solid phase as steric hindrance is reduced. Elimination of Fc-associated effector functions. Simpler system for studying structural basis for immune recognition using X-ray crystallography or NMR. Lower immunogenicity than intact antibody.

Question 13

Why cut out the IgG chain?

Answer 13

Reduce site of antibody make it lighter so antibody can travel further.

Question 14

What is papain?

Answer 14

Thiol-type protease. Cuts 2 separate but identical Fab fragments each with 1 binding site and 1 Fc fragment.

Question 15

What is pepsin?

Answer 15

Acid type protease. Cuts 1 Fab fragment with 2 covalently linked Fab fragments which are bivalent and can still cross link.

Question 16

Compare monyclonal and polyclonal antibodies.

Answer 16

Monyclonal reactive to single epitome. Polyclonal more likely to detect epitome so less antibody needed however is less specific.

Question 17

What are the 5 classes of antibodies?

Answer 17

IgA, IgD, IgE, IgG and IgM. Major class in blood is IgG. 4 subclasses of this.

Question 18

What are polyclonal antibodies?

Answer 18

Whole sera of immunised animal antibodies are purified off other circulating antibodies and contain a mixture of monoclonal antibodies.

Question 19

What are monoclonal antibodies?

Answer 19

An antibody secreting cell isolated from an immunised animal and fused with B-cell tumour. Hybridoma maintained in vitro and secrete antibodies within a defined specificity. Each mAb is immunoreactive to single epitope.

Question 20

What is peroxidase anti-peroxidase PAP method?

Answer 20

Enzyme linked horseradish peroxidase chromogenic substrate that changes colour if reacted with DAB alkaline phosphatase. Light microscopy.

Question 21

Why block endogenous peroxidase activity?

Answer 21

Some cells or tissues contain endogenous peroxidase. Using HRP conjugated antibody may result in high non specific background staining. Can be reduced by pre treatment of cells with hydrogen peroxide.

Question 22

What chemicals block endogenous peroxidase activity?

Answer 22

H2O2.

Question 23

What is the heat induced epitope reversal procedure for?

Answer 23

To reverse loss of antigenecity that occurs with some epitopes. Require antigen recovery. Always required for parafin tissues. Deparaffinaze slides. Wash, add citrate buffer or Tris. Place on hot plate. Rinse with PBS. start immunostaining protocol.

Question 24

What is the difference between epifluorescence and confocal microscopy?

Answer 24

Epifluorescence is cheap, short acquisition time, up to 20micrometre section. Photobleaching.

Question 25

What are images from epifluorescence like?

Answer 25

Light is far from focal plan and provides limited optical resolution. No effect on signal intensity for measurement of co localisation. Max 60micrometre resolution.

Question 26

What are images from confocal like?

Answer 26

Light produced by fluorescence is close to focal plane so excellent resolution of more than 60-100micrometres.

Question 27

What is in situ hydbridisation for?

Answer 27

Detecting sites of gene expression or tissue location of mRNA or viral infections. Localisation of nucleic acid sequences, DNA in chromosomes, RNA in cells.

Question 28

What is FISH?

Answer 28

Fluorescent IN SITU hybridization. Technology utilizing fluorescently labelled DNA probes to detect or confirm gene or chromosome abnormalities. Sample DNA is first denatured. Fluorescent labeled probe of interest is added to denatured sample mixture and hybridizes with the sample DNA at the target site as it reanneals back into a double helix. DNA is scored for presence or absence of signal.

Question 29

What is SKY spectral karyotyping human?

Answer 29

Human chromosomes visualised by painting each pair of chromosomes in a different fluorescent colour. Can detect chromosome abnormalities. Translocation can be seen.