HTHSCI 1DT3 Lecture Notes - Lecture 9: Central Nervous System, Chromosome, Immunoglobulin G
23 Jun 2018
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Signal Amplification
To amplify signals further, one can use biotinylated secondary antibodies and then
fluorophore-coupled streptavidin, which has multiple binding sites for biotin.
Practical Steps for Immunocytochemistry:
Fixation
Live Cells
Permeabilisation
Blocking Non-Specific Binding
Co-immunocytochemistry
Pratical
Examples
Cells are usually immunostained after Fixing
‘Fixing’ cross-links proteins at the cell surface, e.g. paraformaldehyde cross links lysine
residues. !!!
When cells are fixed they die. Fixing ‘fixes’ them just as they were the moment before the
fixing chemical was added.
Commonly used fixatives include: Paraformaldehyde, Formaldehyde, Glutaraldehyde
Some antibodies do not recognise an antigen after it is fixed, and these have to be used on live
cells (if the antigen is on the cell surface)
Cells can be stained LIVE
Antibody binds to (a) cell surface antigen (b) in its native state.
Temperature?
Most common are RT, 4 deg C, 37 deg C. NB: at 37 deg C, and even RT, allow
clustering, patching, capping and endocytosis to occur,
i.e. the antigen can change its location as a consequence of antibody binding
At 4 deg C the antigen is immobile, so the distribution revealed by antibody binding
should be a true reflection of actuality.
Medium? !
Should be buffered appropriately (especially if at 4 deg C or RT), so might need to
add 20 mM Hepes.
Need to be very gentle or cells will be washed off.
If the antigen is inside the cell (intracellular), cells will need to be PERMEABLISED
This punches small holes in the membrane, allowing free access to the cell interior
Methanol
Triton-X100 (detergent, 0.2%)
(Permeablisation is obviously not compatible with LIVE staining, as the cells would die on
permeablisation.
Only cell-surface antigens can be stained live)
Non-specific binding sites should be blocked by incubating cells with medium/PBS containing protein
such as:
Foetal Calf Serum, FCS (eg. 10%)
Bovine Serum Albumin, BSA (e.g. 5%)
Without this step, background binding of your antibody may be high, preventing the discerning of
specific binding to the target antigen.
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Co-immunocytochemistry
The relative distribution of two or more antigens can be determined:
Primary antibodies against each antigen must be raised in animals of different species
…or in the same animal, but be different isotypes, e.g. co-immunocytochemistry can be carried
out using two mouse monoclonal antibodies, as long as one is an IgG isotype and the other
an IgM isotype.
Different secondary antibodies can be used that recognise these different primary antibodies.
Each of the secondary antibodies must be coupled to a different fluorophore.
(also can use biotin-streptavidin)
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Document Summary
To amplify signals further, one can use biotinylated secondary antibodies and then fluorophore-coupled streptavidin, which has multiple binding sites for biotin. Fixing" cross-links proteins at the cell surface, e. g. paraformaldehyde cross links lysine residues. Fixing fixes" them just as they were the moment before the fixing chemical was added. Some antibodies do not recognise an antigen after it is fixed, and these have to be used on live cells (if the antigen is on the cell surface) Antibody binds to (a) cell surface antigen (b) in its native state. Most common are rt, 4 deg c, 37 deg c. nb: at 37 deg c, and even rt, allow clustering, patching, capping and endocytosis to occur, i. e. the antigen can change its location as a consequence of antibody binding. At 4 deg c the antigen is immobile, so the distribution revealed by antibody binding should be a true reflection of actuality.
