Fluorescence microscopy of tumor cell apoptosis
Fluorescence microscopy of tumor cell apoptosis
Fluorescence microscope (Fluorescence microscope): Fluorescence microscope is a light source of ultraviolet light, used to irradiate the object to be examined, so that it emits fluorescence, and then observe the shape of the object and its location under the microscope. It is used to: (1) study the absorption and transportation of substances in cells, and the distribution and localization of chemical substances. (2) There are some substances that cannot fluoresce by themselves, but if they are stained with fluorescent dyes or fluorescent antibodies, they can also fluoresce by ultraviolet light irradiation, and fluorescence microscopy is used to conduct qualitative and quantitative research on such substances.
Operation method
fluorescence microscopy
Principle
1. Apoptosis is the genetically controlled, autonomous and orderly death of cells to maintain the stability of the internal environment. Apoptosis is different from cell necrosis, apoptosis is not a passive process, but an active process, which involves a series of gene activation, expression, and regulation, etc. It is not a phenomenon of self-injury under pathological conditions, but a death process actively striving for better adaptation to the survival environment. 2. Apoptosis is different from cell necrosis, which has a series of cellular morphology and biochemical changes, including the appearance of chromosome condensation, DNA degradation, and the formation of apoptotic vesicles. In normal living cells, phosphatidylserine (PS) is located on the inner side of the cell membrane, but in apoptotic cells, PS is flipped from the inner side of the cell membrane to the surface of the cell membrane and is exposed to the extracellular environment, Annexin-V (membrane-bound protein-V), a Ca2+-dependent phospholipid-binding protein with a molecular weight of 35-36 KD. can bind to PS with high affinity. Therefore, Annexin-V was labeled with fluorescein (e.g., FITC, PE) or biotin, and the labeled membrane-associated protein-V was used as a probe to detect the occurrence of apoptosis by fluorescence microscopy.
Materials and Instruments
Tumor cells Move 1. Collect about 106 cells and wash them twice with cold PBS. 2. 4% formalin was fixed at 4℃ for about 10min and then coated and dried. 3. Stained with 5-10μg/mL hocchst 33258 for 5-10min, rinsed with water, dried and observed with UV (340nm) excitation of hocchhest. Caveat 1. The whole process of the experiment should be as gentle as possible, do not blow the cells, and try to operate at 4 ℃. 2. Detect the cells as soon as possible after the reaction, because apoptosis is a dynamic process, and the fluorescence intensity will start to decay after one hour of reaction. Common Problems The difference between fluorescence microscope and other microscopes: 1, light source: ordinary microscopes use ordinary natural light, while it uses a short wavelength, high resolution ultraviolet light. 2, the illumination method: fluorescence microscope uses a more special falling type, the light source is directly projected on the sample. 3, filter: in order to protect the eyes of the observer, fluorescence microscope in the eyepiece and objective lens installed between the filter can filter out the eyes of the ultraviolet light. When using a fluorescence microscope: 1, to strictly follow the instructions of the instructions for the operation, can not step or arbitrarily switch to change the order. 2、Observation should be done in a dark space. Wait until your eyes have adapted to the dark environment before making observations. 3, in order to avoid ultraviolet damage to the eyes, adjust the light source need to bring protective glasses. 4、Since the light source has a certain service life, all steps need to save time so as to protect the light source and maximize its service life. For more product details, please visit Aladdin Scientific website.
Formalin PBS Ethidium bromide Rhodamine Water
Slides Coverslips Fluorescent probes UV spectrophotometer
Sourced from "Fluorescence Microscopy Morphology and Proliferation Inhibition Study of Apoptosis Induced by 153Sm-EDTMP in Osteosarcoma Cells", Nuclear Technology, Zhu Shoupeng 1999,22(5).