Introduction to Immunofluorescence Technology

PriCells- immunofluorescence (Immunofluorescence technique)

In 1941, Coons was successful in marking with fluorescein for the first time. This technique of labeling antibodies with a fluorescent substance for antigen localization is called a fluorescent antibody technique. The main features of this technology are: high specificity, high sensitivity and fast speed. The main disadvantage is that the problem of non-specific staining has not been completely solved, and the objectiveness of the judgment is insufficient, and the technical procedures are still complicated.

First , the concept of fluorescent immune technology:

Immunofluorescence technique, also known as fluorescent antibody technology, is one of the earliest developments in marker immunology. The antigen or antibody is labeled with fluorescein, and the labeled antigen or labeled antibody is reacted with the corresponding antibody or antigen to determine the fluorescein in the complex. This immunological technique is called immunofluorescein technology. Immunofluorescence cytochemistry is divided into direct method, sandwich method, indirect method and complement method.

Second , the classification of fluorescent immune technology:

(1) Fluorescent antibody microscopy technique: A method for detecting a result by fluorescence microscopy after an antigen-antibody reaction.

(2) Immunofluorescence measurement technology: A method for measuring the concentration of a test substance by measuring the fluorescence intensity by using a special instrument after the antigen-antibody reaction

Third , the production of fluorescence :

The substance absorbs the external energy and enters the excited state. When the ground state is restored, the excess energy is released in the form of electromagnetic radiation, that is, luminescence. This kind of light is called fluorescence. This substance is called fluorescein. Fluorescence caused by photoexcitation is photoluminescence; fluorescence caused by chemical reactions is chemiluminescence.

Fourth, the fluorescein fluorescence characteristics:

(1) The energy supply is stopped and the fluorescence phenomenon is terminated.

(2) The absorption of light and the emission of fluorescence are highly selective. The wavelength of the incident light <the wavelength of the emitted light

(3) Fluorescence efficiency: fluorescence efficiency = photon number of emitted fluorescence / photon number of absorbed light

(4) Fluorescence quenching: the radiant ability of fluorescein is weakened

Five , common fluorescein :

(1) Fluorescein isothiocyanate (FITC): FITC pure product is a yellow or orange-yellow crystalline powder, easily soluble in water and alcohol solvents. There are two isomers, of which isomer type I is superior in terms of efficiency, stability and protein binding. The FITC has a molecular weight of 389.4, a maximum absorption wavelength of 490 to 495 nm, and a maximum emission wavelength of 520 to 530 nm, which exhibits bright yellow-green fluorescence. FITC can be stored in cold and dark dry places for many years and is the most widely used fluorescein. The main advantage is that the human eye is more sensitive to yellow-green, and the green fluorescence in the sliced ​​specimen is usually less than red.

(2) tetraethyl rhodamine (RB200): RB200 is an orange-red powder, insoluble in water, soluble in alcohol and acetone, stable in nature and long-term storage. The maximum absorption light wavelength is 570 nm, and the maximum emission light wavelength is 595 to 600 nm, showing orange-red fluorescence.

(3) tetramethyl rhodamine isothiocynate (TRITC): TRITC is a derivative of rhodamine, which is a purplish red powder and is relatively stable. The maximum absorption light wavelength is 550nm, the maximum emission light wavelength is 620nm, showing orange-red fluorescence, which is contrasted with FITC's emerald green fluorescence, and can be used for double labeling or contrast dyeing. Due to its slow fluorescence quenching, it can also be used for separate labeling staining.

(4) Lanthanide: lanthanide chelates Some trivalent rare earth lanthanides such as europium (Eu3), strontium (Tb3), cerium (Ce3) and the like can also emit characteristic fluorescence after being excited. Among them, Eu3 is the most widely used. The Eu3 chelate has a wide range of excitation light wavelengths, a narrow wavelength range of emission light, and a long fluorescence decay time, which is most suitable for the resolution of fluorescence immunoassays.

(5) P-phycoerythrin (PE): PE is a natural fluorescent pigment found in red algae. It has a molecular weight of 240kD and a maximum absorption peak of 564 nm. When using 488 When the nm laser is excited, its emission fluorescence peak is about 576 nm. For a single-laser flow cytometer, a 585±21 nm band-pass filter is recommended. For a dual-laser flow cytometer, a 575±13 nm band is recommended. Pass filter. The FL2 detector detects the PE.

(6) Peridinin chlorophyll protein (PerCP): PerCP is found in the optical synthesizer of Dinoflagellate and Dinoflagellate. It is a protein complex with a molecular weight of about 35kD and a maximum excitation wavelength peak at 490nm. Nearby, when excited by a 488 nm argon ion laser, the peak of the emitted light is about 677 nm. The FL3 detector detects PerCP.

(7) Propidium iodide (PI): can be selectively inserted into a double-stranded base pair of nucleic acids (DNA, RNA). When staining DNA, cells should be treated with RNase to rule out the effect of RNA on the accuracy of DNA fluorescence quantification. The excitation spectrum of PI is 610-620 nm under excitation at 488 nm. The FL2 detector detects PI.

Common fluorescein properties:

(1) FITC: yellow crystalline powder, absorbed light: 490~495nm, emitted light: 520~530nm, bright yellow-green fluorescence.

(2) RB200: orange-red powder, absorbing light 570 nm, emitting light 595-600 nm, orange-red fluorescence.

(3) TRITC: purple red powder, absorbing 550 nm, emitting light 620 nm, orange-red fluorescence.

(4) 镧: Eu, Tb

(5) PE: absorption light 490 ~ 560nm, emission light 595nm, red fluorescence.

(6) Others: A fluorescent substance is produced after the action of the enzyme.

The enzyme produces a fluorescent substance:

Some substances that produce fluorescence after enzymatic action Some compounds have no fluorescence effect, and once they are enzymatically formed, they form a substance with strong fluorescence. For example, 4-methylumugal-β-D-galactoside is decomposed into 4-methylumbelliferone by the action of β-galactosidase, which emits fluorescence with an excitation wavelength of 360 nm and an emission wavelength of 450 nm. . Other substrates such as alkaline phosphatase 4-methylumbellone phosphate and horseradish peroxidase substrate p-hydroxyphenylacetic acid and the like. Lanthanide Chelate Certain chelates of rare earth lanthanides such as europium (Eu3+) and strontium (Tb3+) emit characteristic fluorescent light, and have a wide range of excitation light wavelengths, narrow wavelength range of emitted light, and fluorescence decay. Long time, it is most suitable for time-resolved fluorescence immunoassay.

Sixth , the choice of suitable fluorescein :

(1) A chemical group having a covalent bond with a protein.

(2) The fluorescence efficiency is high, and the decrease after labeling is not obvious.

(3) Fluorescent color and background color contrast.

(4) Maintaining biological activity and immunological activity after labeling

(5) The marking method is simple and fast.

(6) Safe and non-toxic