透射电镜 z-bo 2020-08-13 522

　　Main reagent

　　glutaraldehyde (imported by EMS, special for electron microscope) PFA PBS or PB buffer 1-4% osmium tetroxide in buffer 0.5% uranyl acetat ethanol epoxypropane resin

　　Main equipment fume hood electron microscope

　　Experimental steps

　　1. Material: Prepare the required instruments and fixative in advance, and put them in the refrigerator at 4℃ for pre-cooling. The tracheal and eye lens should be quickly rinsed with normal saline or directly put into the pre-cooled 2.5% glutaraldehyde fixative (the shelf life is 1 month) after being isolated, and store it in the refrigerator at 4°C , Generally required to be completed within two minutes. When handling surgical resections, two sharp blades (such as razors or scalpels) and wax plates should be prepared in advance, and the fixation liquid should be dropped on the wax plates, and the cut organs should be cut into small pieces with a scalpel. Block, put it in the fixative on the wax board, and then use the double-blade sawing method (please consult the staff of our office) in the fixative to cut the tissue into about 1 cubic millimeter in size or about 1 square millimeter in cross section Long strip, use scissors to make a small opening for the 1ml pipette tip, and then gently suck a small piece of 1 cubic millimeter into the EP tube containing the fixing solution (preferably a 2ml round-bottomed EP tube, so that it can be fully immersed in the fixed Liquid to make the fixation sufficient). Make a mark, put it at 4℃, change the fixative after 1h, then put it at 4℃, it can be stored for one week.

　　2. Second fixation: Wash 5 times*10 times with PB, then fix with 1% osmium tetroxide for 1h;

　　3. Dyeing: ddH2O washing 5 times*10min, uranium dyeing 2h;

　　4. Dehydration: 30%ethanol 50%ethanol 70%ehanol (can be overnight at 4℃) 90%ethanol one time for 15min and then 100%ethanol 10min*2 times epoxypropane 10min*2 times;

　　5. Soaking: 1part resin/1part solvent(ddh2o) 3parts resin/1part solvent in turn for more than 2h-overnight 100% resin for more than two hours or overnight room temperature;

　　6. Embedding: In order to accelerate the solidification rate of resin, add catalyst DMP-30, 35ul to 2ml resin, first soak the resin with DMP-30 for about 2h, and then embed. The embedding is carried out in the embedding plate. The resin containing the catalyst is added to each small hole in the embedding plate, and then a small piece of tissue is picked out with a toothpick and placed on one end of the small hole, and the other piece is picked out and placed At the other end, each one is made in parallel. Then put the embedding plate in a 60 ℃ oven for 48 hours or more to solidify the embedding block.

　　Precautions

　　1. Precautions for sampling: The key to the preparation of biological samples for electron microscopy is that the prepared samples must faithfully reflect the state of their lives, and try to avoid "post-mortem changes" and artificial artifacts. Therefore, the step of sampling is very important. The requirements for obtaining materials are as follows: 1) Cold: the equipment and fixative used for obtaining materials should be pre-cooled in a refrigerator at 4℃. 2) Fast: When taking the material, the action should be fast, and the blade used should be sharp. Generally, the material should be taken within 1-2 minutes after the organ is separated and stored in a 2.5% glutaraldehyde fixative solution in a refrigerator at 4°C. In addition, squeezing and artificial damage to the tissue should be avoided. 3) Small: In order to make the tissue fully fixed, the tissue must be cut into as small pieces as possible. Generally, the tissue piece is required to be about 1 cubic millimeter or the cross section is a long strip of 1 square millimeter, and the length generally does not exceed 3 mm. The skin, tendon and other tissues are dense, and the material should be within 1 cubic millimeter. 4) Accuracy: As electron microscopy specimens require small pieces of material, for the accuracy of electron microscopy observations, the location of the material must be accurate, and the specimens of different experimental groups should be taken as far as possible in the same position (such as myocardial tissue from the left ventricle, the left ventricle should be taken. ), otherwise, it will not be able to compare, which will affect the observation results and eventually lead to the failure of the electron microscope observation. 5) Different tissues may require different fixatives, such as 2.5% glutaraldehyde 2% PFA for trachea and 2.5% glutaraldehyde for eyes.

　　2. During the process of dehydration, soaking and washing, don't let the tissue block get out of liquid, so as not to dry out. Osmium acid is highly toxic and should be operated in a fume hood. Uranium is radioactive and should also be in a fume hood, and the tips used during the operation should be handled in a centralized manner.