Lecture 2 - Microscopy 2

Question 1

Immunofluorescence

Answer 1

direct : tissue has an antigen and a fluoresceintated antibody binds to it and emits light
indirect : tissue has an antigen, an antibody 1 binds to it, and a fluoresceinated anti-antibody 1 binds to the antibody 1 and emits light.

Question 2

immuno fluorescence procedure

Answer 2

tissues are usually sectioned prior staining
antibody cannot cross if cells are alive, we fix the cells with formaldehyde (or methanol or gluteraldehyde), then permeabilize cells with detergent (can be skipped if you stain the surface of cells). Add the primary antibody, and if needed o the secondary antobdy.

Question 3

antibodies and colors used together

Answer 3

different colours can be attached to different antibodies.
several proteins can be tracked at once. when we don’t knoe the localization of a protein, we can compare it to known proteins in one cell.
usually 3 colour are used toegther, can be be more.

Question 4

making antibodies

Answer 4

they are produced by B-lymphocytes and plasma cells. we usually use IgG antibodies to analyse cells.
Y-shaped molecule with 2 light chains and 2 heavy chains, and two antigen binding sites, and the tip of each arm of the Y (N-terminal)
the antigen which binds the antibody is called the epitope
antibodies can be created against most macromolecules

Question 5

antibodies for immunocytochemistry

Answer 5

immunisation: animals are injected at specific interval with suspension of the antigen conjugated or not. at the end blood is removed from animal and the serum is separated and test by immunocytochemistry

if a satisfactory immunostaining is obtained, the new antibody is characterized to make sue in recongizes the antigen with specificity.

Question 6

Monoclonal antibodies

Answer 6

recognize only one epitope and are the product of a hybrid myeloma cell line (fusion of myeloma and lymphocytes from an animal immunized with antigen), called Hybridomas. they grow easily and indefinitly in tissue culture
myeloma cell lines used for antibody production are azaguanine-resistant, cannot survide in a medium called HAT.
monoclonal antibodies only from rats and mice

Question 7

preparation of monoclonal antibodies

Answer 7

1. rats/mice are imunised with antigen
2. animals are bled and sera tested by immunocytochemistry
3. animals that produce the best polyclonal antibodies are selected for fusion and further immunised
4. at the end of immunisation, lymphocytes from the animal are fused with the myeloma cells
5. eliminate non-fused cells: cells are grown in HAT medium, killing the original myeloma cells.
6. fused cells are grown in wells of tissue culture plate in a normal medium
7. the antibody production is tested from the spent culture supernatant from each well.
8. at first each well has more than one clone of hybridoma
   9.hybridoma cells are plated in wells so that an avergage of one cell per well is obtained

Question 8

advantages of monoclonals

Answer 8

can be obtained in high amounts at low cost
hybridoma are immortal cell lines
monoclonals produce highly specific staining

Question 9

microinjections

Answer 9

precise and localized delivery of substances like fluorescent dyes, tracers or molecules into cells, to track image or monitor their activity and fate. this technique can be automated allows for high throughout labeling at single-cell resolution

Question 10

Green Fluorescent Protein

Answer 10

GFP fusion proteins are ideal for use in time-lapse imagine and photbleaching experiments in living cells.
1. plasmid grown in bacteria
2. purify the plasmid with the GFP protein
3. transfect mammalian cells
4. GFP-protein fusion is expressed after 24h
5. examine living cells on heated microscope stage

Question 11

making GFP-tagged proteins

Answer 11

GFP-tagging proteins involves creating a new artifical gene in which the DNA coding sequence for GFP is fused to the protein
This gene is usually created on a bacterial plasmid and must be introduced into the cell
thus GFP is much easier to use in tissue culture cells than in animals

Question 12

advantages of GFP

Answer 12

• can be used in both living and fixed cells
• if DNA is introduced into cell, GFP-tagged protein is produced by cell and already fluorescent
• good for live imaging
• can now be used with other colors of fluorescent protein or even in combination with immunofluorescence.

Question 13

disadvantages of GFP

Answer 13

sometimes GFP fusion protein does not fold properly or otherwise misbehaves
protein may be present in cell in higher than physiological amounts
endogenous protein in cell is not visualized
expression of GFP-proteins in whole animals is much more difficult than in tissue culture cells.