Immunofluorescence Microscopy

What is immunocytochemistry?

The use of antibodies to locate antigenic molecules in cells and tissues.

What is immunofluorescence (IF)?

The use of antibodies that have been conjugated to fluorescent molecules called fluorochromes.

What are the two types of immunofluorescence?

Direct (using only one antibody type) and indirect (using a non-fluorescent primary followed by a fluorescent secondary).

What is the characteristic function of a fluorochrome?

Every fluorochrome has a characteristic energy of light it can absorb and a characteristic energy or wavelength that the photon emits.

At what wavelength does fluorescein isothiocyanate (FITC) absorb and emit light?

FITC absorbs at 490nm and emits at 525nm, typically appearing green.

At what wavelength does rhodamine (TRITC) absorb and emit light?

TRITC absorbs at about 560nm and emits at 610nm, typically appearing orange-red.

What is a fluorescence microscope?

A microscope that has a special light source and filters, producing UV excitatory wavelengths, usually from a high-power mercury vapor lamp or LEDs.

What is photo-oxidation/photobleaching?

The process where some excited fluorochromes return to their ground state by transferring lost excited electrons, making them incapable of emitting fluorescent photons.

What are quenching agents?

Agents that inhibit photobleaching and are liquids in which labeled cells are bathed.

What must cells examined by immunofluorescence do to allow antibody access?

Cells must be permeabilized, which usually results in cell death.

What does fixation do in the context of immunocytochemistry?

Fixation preserves the cell structure so it does not change after the cell is killed.

Which agents are commonly used for fixation?

Formaldehyde and glutaraldehyde, which form covalent cross-links between proteins.

How must membranes of aldehyde-fixed cells be treated?

They must be permeabilized using detergents like Triton X-100 or incubation in ice-cold methanol.

What role does BSA play in the immunocytochemistry process?

BSA is used as a blocking agent in the procedure, followed by rinsing with PBS.

What type of secondary antibody is mentioned in the notes?

Rabbit polyvalent anti-mouse Ig, FITC-conjugated.