Classification and usage of fluorochromes
Labeling of proteins, nucleic acids, saccharides, lipids and toxins
Fluorescence-labeled nucleotide: Cy3 labeled dCTP
Used in labeling of FISH probes (by means of Nick translation*).
Fluorescence-labeled antibodies: FITC-labeled anti-mouse IgG (or IgG & M) antibodies
Antibody (Secondary antibody) against IgG or IgM (Primary antibody) from various animals is labeled with fluorescent dyes.
Fluorescence-labeled avidin: Texas Red-labeled streptavidin (or avidin)
This probe can be used to detect the biotin-labeled FISH probe or antibody because the avidin can bind specifically with the biotin.
Fluorescence-labeled lipid: NBD-labeled phosphocholine
Lipid labeled with the fluorescent dye is mixed with the membrane lipid to measure the membrane fluidity by means of photo-bleaching (FRAP), and to observe the process of membrane fusion.
Fluorescence-labeled dextran: Texas Red-labeled Dextran
Dextran, a saccharide with a high molecular weight of 3,000 to 70,000, is labeled with a fluorescent dye or probe (calcium ion probe, pH probe, fluorescein, etc.). It is injected into a cell so that it is distributed uniformly in the cytoplasm.
Fluorescence-labeled phallotoxin: Rhodamine phalloidin
Bundles of actin filaments (a stress fiber) can be observed in cells because the phalloidin and the phallacidin can bind specifically with actin filaments (microfilaments).
Fluorescence-labeled agonists/antagonists: TRITC-labeled alpha-bungarotoxin
The distribution of receptors can be observed at neuromuscular junctions because the alpha-bungarotoxin binds specifically with acetylcholine receptors.
The nick translation is a method of labeling DNA fragments for FISH probes. The reagents are usually provided in the form of a kit. The DNA catabolic enzyme (DNase 1) nicks randomly one of the two DNA strands in the primary reaction, and the second enzyme (DNA polymerase 1) digests the DNA from the nick and simultaneously synthesizes DNA at the digested portion. An expanse of newly synthesized DNA contains fluorescent dye-labeled nucleotides. The biotin nucleotide (biotin-16-dUTP) or digoxigenin nucleotide (dioxigenin-11-dUTP) can be used in place of the fluorescent dye-labeled nucleotide.
Staining of lipid membrane
Membrane potential-sensible dyes: WW 781, RH-155, Di-8-ANEPPS
These dyes are incorporated into the cell membrane. Their absorption or fluorescence intensity varies depending on the membrane potential. Fast-type dyes with response rates on the order of milliseconds should be used.
Organelle membrane-specific dyes: DiOC6(3), NBD ceramide, Rhodamine 123
These dyes stain specifically the organelle membranes, such as the endoplasmic reticulum membrane, golgi membrane and mitochondrial membrane.
Tracer dyes: DiI, DIO, DIA
These dyes are incorporated into the cell membrane and are used in the observation of nerve extension.
Staining of cytoplasm
Ion concentration-sensible dyes: Fura-2, Indo-1, Fluo 3, BCDCF, c SNARF-1
Membrane-permeable AM (acetoxymethyl) ester of the dyes is introduced into cells easily. Their fluorescence intensities and excitation/fluorescence spectra vary depending on the intracellular ion concentration (calcium ion, pH).
Tracer dyes: Sulforhodamine 101, Lucifer Yellow CH
These water-soluble dyes are micro-injected into the cell for the observation of the extension of nerve cells.
Live cell staining dyes: Calcein AM, CFDA (Carboxyfluorescein Diacetate)
Esterases in cells turn the membrane-permeable dyes into membrane-impermeable dyes, thereby trapping them inside the cells.
Staining of nucleic acids: DAPI, Propidium lodide, Hoechst 33342, YOYO-1
These dyes bind with nucleic acids (DNA, RNA) inside the cell and become fluorescent. They are used to observe the morphology of nuclei and chromosomes and to determine DNA quantity.
Fluorescent proteins: GFP (Green Fluorescent Proteins)
This protein has been found in the jellyfish and emits green fluorescence under blue-light excitation. The GFP gene has been isolated and improved to get different color variants.
Fluorescent enzyme substrate: Dihydrorhodamine 123
The activity of an enzyme can be measured on a microscope as the dye becomes fluorescent by the specific enzyme in the cells.
Fluorescent latex: FluoSpheres (Molecular Probes, Inc.)
Polystyrene beads 0.01 to 15 micrometers are used in the observation of the transport in axons, research into phagocytosis, and measurement of blood flow, or as the standard for fluorescence microscopy.
