Lab 2: Immunofluorescence Flashcards
What are the differences between fluorescence microscope and super resolution laser scanning confocal microscope?
- Fluorescence microscope uses LED reflected light while LSM uses lasers on the sample.
- Fluorescence microscope resolution is up to 300nm while LSM goes up to 120nm.
What does ‘resolution’ mean?
It is the shortest distance that can be measured between to points or structures which are considered as separate entities
Why do we need to use Immunofluorescence?
In order to visualize sub cellular structures and specific organelles under the microscope, we need to use immunofluorescence.
To see DNA, organelles (ex: mitochondria, ER etc.) or specific proteins (ex: actin filaments or microtubules, etc.) inside the cell, we need to a technique called Immunofluorescence (IF)
What reaction does the Immunofluorescence technique rely on?
The antibody-antigen reaction where we use fluorophores on the antibody that give a certain color based on the wavelength of the fluorophore used.
In order to see the colour, what type of light does the fluorescence microscope use?
Reflected light instead of transmitted light
What is Immunofluorescence?
It is a technique that utilizes the highly specific binding of antibodies to their target antigens (or protein targets) as a way of visualizing materials and structures inside the cell.
What are antibodies? How are they produced? Structure?
Antibodies are large complex proteins produced by the B cells (specific immune cells) in response to the presence of a foreign agent in the body, recognized by the immune system.
They are composed of 4 peptide chains, 2 heavy, 2 light.
What determines the differences in antibodies?
At the ends of the “arms” of the ‘Y’ is a small, highly variable region made up of a region of the light and heavy chains. Changes in the shape of this region determines the differences between antibodies.
The area on the antigen which binds to the ‘variable region’ on the antibody is called?
Epitope
What is used to wash the cells in IF?
PBS (Phosphate buffered solution) , a buffer solution that is isotonic and maintains pH level of around 7.2.
What is “fixation”?
The preservation process by ‘freezing’ the proteins in place by a chemical process. We use formaldehyde (or paraformaldehyde) which forms permanent covalent cross links between protein chains and locks proteins into their structure, preserving the structure of the cell.
What is “permeabilization”? Why is it done?
It the process by which holes are made into the plasma membrane in order to accommodate for the entrance of the large protein molecules (antibodies during Immunofluorescence) across the membrane.
We use Triton X100 ,a detergent, which works by solubilizing lipids in water.
What is “blocking”?
Blocking is the addition of a solution of proteins that soaks up the non-specific binding sites, so that when we add the antibodies, they can bind only to their specific target sites.
We use Bovine Serum Albumin (BSA) for blocking.
What is the antibody used?
Anti human actin 488. It is an antibody that recognizes actin from human and has a color green.
What is DAPI?
It is a fluorescent dye that stains the DNA in the nucleus blue (405 nm)
