Immunofluorescence Microscopy

Immunocytochemistry -> the use of antibodies to locate antigenic molecules in cells and tissues 

Immunofluorescence/IF -> use of antibodies that have been conjugated to fluorescent molecules called fluorochromes  

• Can be direct if only one antibody type is used or indirect if a non-fluorescent primary is used followed by a fluorescent secondary 

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

• Fluorescein isothiocyanate (FITC) absorbs at 490nm and emits at 525nm, typically a green color 

• Rhodamine (TRITC) absorbs at about 560nm and emits at 610nm, typically an orange-red color 

Fluorescence microscope -> has special light source and filters, UV excitatory wavelengths are produced by a high-power mercury vapor lamp or LED’s 

• Light is directed from above 

Photo-oxidation/photobleaching -> some of the excited fluorochromes return to their “ground state” because they lose their excited electrons by transferring to other molecules, they are then no longer capable of being excited and emitting a fluorescent photon 

Quenching agents -> serve to inhibit photobleaching, liquids in which labeled cells are bathed 

Cells examined by immunofluorescence must be permeabilized in order to allow large antibody molecules to have access to the cell’s interior 

• Can’t be done without killing the cell 

• Fixation -> fixing aspects of the cell structure so they don’t change after killing the cell 

• Formaldehyde and glutaraldehyde often used, form covalent cross-links between and within proteins 

• Membranes of aldehyde-fixed cells must be permeabilized using detergents (Trition X-100) 

• Can also incubate cells in ice-cold methanol which precipitates proteins in place 

• Don’t require further permeabilization 

BSA used as a blocking agent in this procedure, rinsing with PBS 

Secondary antibody -> rabbit polyvalent anti-mouse Ig, FITC-conjugated