Fluorescein commonly used in immunofluorescence histochemical staining are: fluorescein isothiocyanate (FITC), tetramethyl rhodamine isothioeyanate (TRITC), tetraethyl rhodamine (tetraethyl rhodamine) B200, RB200), Texas red (Texas red), phycoerythrin (PE), cyanines (cyanine, such as Cy3, Cy5), etc. In addition, there are some new fluorescent dyes, such as quantum dots.
(1) Fluorescein isothiocyanate (FITC): FITC has stable properties, is easily soluble in water and ethanol, and can bind to proteins. It is the most commonly used fluorescent probe for detecting proteins in tissue cells. It can also label antibodies and can be used for single or multiple staining in immunohistochemistry. The disadvantage is that it is easily quenched under light and is easily affected by autofluorescence. The maximum excitation light wavelength is 490nm; the maximum emission light wavelength is 525nm, showing yellow-green fluorescence.
(2) Rhodamine Tetramethylisothiocyanate (TRITC): This fluorescein can bind to intracellular proteins, has better stability than FITC, is insensitive to pH changes under physiological conditions, and has little fluorescence intensity due to autofluorescence interference . The maximum excitation light wavelength is 550nm; the maximum emission light wavelength is 620nm, showing orange-red fluorescence, which is in sharp contrast with the yellow-green fluorescence emitted by FITC, and is often used for immunofluorescence histochemical double staining.
(3) Tetraethyl rhodamine (RB200): It can be combined with intracellular proteins, insoluble in water, soluble in ethanol and acetone, stable in nature, can be stored for a long time, and is widely used in double-labeled tracer staining. The maximum excitation light wavelength is 570nm, and the maximum emission light wavelength is 595-600nm, showing orange-red fluorescence.
(4) Cyanine dyes: Cy3, Cy5, etc. are commonly used, which can bind to intracellular proteins. The fluorescent properties of these dyes are similar to those of traditional fluorescein, but they are more water-soluble and photostable, have higher fluorescence quantum yields, and are insensitive to pH and other environments. Often used for multiple staining. Cy3 has a maximum excitation wavelength of 570 nm and a maximum emission wavelength of 650 nm, showing green fluorescence. However, Cy3 can also exhibit red fluorescence under excitation at green spectral wavelengths. Cy5 has a maximum excitation wavelength of 649 nm and a maximum emission wavelength of 680 nm, showing red fluorescence. Since the maximum emission wavelength of Cy5 is 680nm, it is difficult to observe with the naked eye, and a high-pressure mercury lamp cannot be used as an ideal excitation light source. Therefore, Cy5 is not recommended when using an ordinary fluorescence microscope. Usually a laser scanning confocal microscope is used to observe Cy5.
(5) Methyl acetate: It does not fluoresce itself, but after it penetrates the membrane and enters the cytoplasm, it is converted into methyl acetate with fluorescent properties under the action of esterase. Its excitation spectrum is pH-dependent, and it is the most used intracellular pH fluorescent indicator. The maximum excitation light wavelength is 505nm, and the maximum emission light wavelength is 530nm, showing green fluorescence.
(6) Indo-1: It is a typical double emission fluorescent probe. There is an emission peak at around 485 nm without calcium, and an emission peak at 405 nm after calcium is combined. The ratio of the two has a linear relationship with the intracellular free calcium ion concentration. Therefore, this probe can be used to quantitatively detect the concentration of intracellular free calcium ions. The maximum excitation light wavelength is 330/346nm, and the maximum emission light wavelength is 405/485nm, showing purple (405nm) or cyan (485nm) fluorescence.
(7) Quantum dot: It is a new type of fluorescent dye developed in recent years, also known as semiconductor nanocrystal. It is a semiconductor material composed of hundreds or thousands of nanoscale particles with stable properties. Soluble in water, cells cannot synthesize and assemble by themselves. Quantum dots have the advantages of long fluorescence time, multiple colors, convenient detection and wide application range. When a certain wavelength of excitation light irradiates a variety of quantum dots with different sizes, multiple colors can be observed at the same time, so the observation and detection of multiple targets can be performed simultaneously. Quantum dots can be coupled with various molecules such as antibodies and streptavidin to detect the distribution and function of target molecules.