Immunocytochemistry

Question 1

Definition

Answer 1

Method for detecting molecules in tissue sections with microscopy, baed on the use of antibodies.

Question 2

Antigen

Answer 2

Molecule to be detected

Question 3

Antibody

Answer 3

Ig molecule that specifically recognises the antigen

Question 4

Epitope

Answer 4

Specigic part of the antigen that is recognised by an Ig molecule. Relatively small, 5-8 amino acids for a protein.

Question 5

Polyclonal antibody

Answer 5

Inject antigen into animal, wait for immune response to develop, remove blood, make serum (may need to purify to gte rid of other antibodies)

Question 6

Monoclonal antibody

Answer 6

Inject antigen into ajimal, remove b-lymphocytes from spleen, hybridise with myeloma cells (immortalised cell line) making one type of antibody in culture.

Question 7

Fixation

Answer 7

Method depends on experiment; perfusion (for animal tissue), immersion (for human tissue or in vitro animal tissue)
Fixatives; formaldehyde (LM) , formaldehyde + gluteraldehyde (EM)

Question 8

Sectioning

Answer 8

Vibrating monotome
Frozen sections
Embedded in parafin wax
Embedded in resin (EM)

Question 9

Basic principles of immunoreaction

Answer 9

Apply antibody to tissue section (it binds to antigen), subsequent rinsing will remove it from other sites. Some kind of label needs to be attached to antibody so it can be seen by LM or EM

Question 10

Types of label

Answer 10

Fluorescent dyes - fluorescein (green) , rhodamine (red), can reveal more than one antigen with different dyes. Not suitable for EM.
Enzymes - generally HRP, suitable for EM or LM.
Colloid gold particles - usually for EM

Question 11

Antibody detection

Answer 11

Could label the antibody raised against the antigen but seldom done as expensive. Generally indirect approach is used. Unlabelled primary antibody is applied, this is then revealed with secondary antibody, secondarh antibodies can be mass produced cheaply.

Question 12

Multiple labeling

Answer 12

Use two or more primary antibodies raised in different species and reveal these with two or, ore secondary antibodies with different fluorescent labels. Secondary antibodies are specific for Ig from a particular species.

Question 13

EM immunocytochemistry

Answer 13

Electron dense matter is seen (HRP and colloidal gold products)
Tissue must be embedded in resin to allow ultrathin sections to be cut.
Pre-embedding- reaction done on thick sections before tissue is embedded in resin
Post-embedding - reaction carried out on thin sections after embedding in resin.

Question 14

Pre-embedding

Answer 14

Advantage - will work for most antigens

Disadvantage - subcellular localisation not perfect, some spread of reaction products

Question 15

Post-embedding

Answer 15

Advantage - excellent localisation

Disadvantage - not suitable for most antigens, easily damaged during EM processing