Microbial experiment frozen section method and precautions !
1. Preparation before the experiment 1. Clean the test bench and instrument 2. Turn on the cryostat and pre-cool the cryostat to the required temperature (- 15~-20 °C*)
3. Prepare well-treated slides, blades, glue and medicines
Second, the material <br> After dissection, quickly remove the required fresh tissue, dry with dry gauze or filter paper, do not need to fix the direct material (24 × 24 × 2mm *), in order to prevent the formation of ice crystals resulting in different shapes in the slices Cavitation, shifting the intracellular structure.
Note: The target specimen in the material should be clear and ensure the structural integrity, avoid unnecessary fat and necrotic tissue adhesion; try to eliminate hair, hard bone or calcification; breast preservation surgery due to more fat tissue, The thickness of the material should not exceed 3mm. The thicker is time-consuming and the old one is difficult to cut. Thyroid and lymph node tissues should have a tissue envelope and remove excess fat tissue.
Third, frozen section 1. Take out the tissue supporter, lay the tissue flat, drip the embedding agent around, quickly put it on the freezer, freeze it, press the tissue with the heat sink, until the embedding agent and tissue freeze into white ice body Can be sliced ​​(1~3 minutes).
2. Clamp the frozen tissue block onto the slicer holder, start the coarse advance and retract key, and turn the knob to level the tissue.
3. Adjust the thickness to be cut, according to different tissues, in principle, the cell is dense and thin, and the fiber multi-cell thin can be slightly thick, generally between 5 and 10 μm.
4. Adjust the anti-roll plate. The key to making frozen slices is the adjustment of the anti-rolling plate, which requires the operator to carefully adjust and adjust it to the proper position. When slicing, the cut, complete and smooth slice is taken as the standard. When the cut tissue is adhered on a clean glass slide, gently push it along one direction to avoid wrinkles during the tissue sticking process, and ensure the integrity of the tissue structure and the beautiful appearance of the slice.
Note: 1 Selection of embedding agent: embedding agent is an important factor affecting the quality of frozen section. The amount of embedding agent should be suitable. Too much or too little will affect the quality of specimen freezing. Commonly used are three kinds of embedding agent OCT agent, B super chelating agent and ordinary glue. B super chelating agent is suitable for cells with rich cell rich texture. Ordinary glue or OCT agent is suitable for hard fibers with rich texture. (OCT embedding agent solidifies when quenched, its freezing speed and soft and toughness are similar to the structure, it also has water solubility, does not affect the dyeing and so on.)
2 The tissue block is too small: first squeeze a small amount of glue on the bracket and freeze it in the freezer. After about 30 seconds, the small tissue will be placed when the glue is solidified, and some glue will be added around the tissue, and then placed in the freezer. Freezing, so that the tissue is raised, you can quickly cut high-quality slices.
3 The temperature of the freezer and the freezing head should be determined according to different tissues. If the temperature is too low, the tissue block will be too hard, the slice will be broken, and the terrace will be thin and uneven or void; on the contrary, if the temperature is too high, the hardness of the tissue block is not enough, and the slice is not easy to form or wrinkle. Data Figure 1 is for reference.
4 When slicing, if it is found that the freezing is excessive, the frozen tissue can be taken out together with the supporter, left at room temperature for a while, then sliced, or the tissue block is pressed with a thumb, or the tissue block is pressed with a thumb to soften the tissue. Slice again. In addition, raise the freezing point.
5 Slides for attaching slices should not be stored in the freezer and stored at room temperature. Because when the slice is attached, there is a temperature difference between the slide taken from the room temperature and the slice in the freezer. When the temperature of the slide is attached to the lower temperature slice, the temperature between the two substances is The difference, when they collide, the molecules move with each other to create an adsorption force that allows the slice to be firmly attached to the slide. If a refrigerated slide is used to attach the slice, the above phenomenon does not occur because the temperature is the same.
Beijing Baiou Bowei Biotechnology Co., Ltd. has Biolog microbial identification system, ultra-low temperature refrigerator, biological safety cabinet and other equipment to carry out routine molecular experiments on microbial separation and identification. The demand for life science research, biotechnology innovation and industrial development in China is actively facing the society and even collecting and collecting microbial resources. Under the premise of ensuring biosafety and protecting intellectual property rights, we provide microbial species resources, genetic resources, information resources and professional technical services for industrial and agricultural production, health, environmental protection and scientific research.
Http://
1. Preparation before the experiment 1. Clean the test bench and instrument 2. Turn on the cryostat and pre-cool the cryostat to the required temperature (- 15~-20 °C*)
3. Prepare well-treated slides, blades, glue and medicines
Second, the material <br> After dissection, quickly remove the required fresh tissue, dry with dry gauze or filter paper, do not need to fix the direct material (24 × 24 × 2mm *), in order to prevent the formation of ice crystals resulting in different shapes in the slices Cavitation, shifting the intracellular structure.
Note: The target specimen in the material should be clear and ensure the structural integrity, avoid unnecessary fat and necrotic tissue adhesion; try to eliminate hair, hard bone or calcification; breast preservation surgery due to more fat tissue, The thickness of the material should not exceed 3mm. The thicker is time-consuming and the old one is difficult to cut. Thyroid and lymph node tissues should have a tissue envelope and remove excess fat tissue.
Third, frozen section 1. Take out the tissue supporter, lay the tissue flat, drip the embedding agent around, quickly put it on the freezer, freeze it, press the tissue with the heat sink, until the embedding agent and tissue freeze into white ice body Can be sliced ​​(1~3 minutes).
2. Clamp the frozen tissue block onto the slicer holder, start the coarse advance and retract key, and turn the knob to level the tissue.
3. Adjust the thickness to be cut, according to different tissues, in principle, the cell is dense and thin, and the fiber multi-cell thin can be slightly thick, generally between 5 and 10 μm.
4. Adjust the anti-roll plate. The key to making frozen slices is the adjustment of the anti-rolling plate, which requires the operator to carefully adjust and adjust it to the proper position. When slicing, the cut, complete and smooth slice is taken as the standard. When the cut tissue is adhered on a clean glass slide, gently push it along one direction to avoid wrinkles during the tissue sticking process, and ensure the integrity of the tissue structure and the beautiful appearance of the slice.
Note: 1 Selection of embedding agent: embedding agent is an important factor affecting the quality of frozen section. The amount of embedding agent should be suitable. Too much or too little will affect the quality of specimen freezing. Commonly used are three kinds of embedding agent OCT agent, B super chelating agent and ordinary glue. B super chelating agent is suitable for cells with rich cell rich texture. Ordinary glue or OCT agent is suitable for hard fibers with rich texture. (OCT embedding agent solidifies when quenched, its freezing speed and soft and toughness are similar to the structure, it also has water solubility, does not affect the dyeing and so on.)
2 The tissue block is too small: first squeeze a small amount of glue on the bracket and freeze it in the freezer. After about 30 seconds, the small tissue will be placed when the glue is solidified, and some glue will be added around the tissue, and then placed in the freezer. Freezing, so that the tissue is raised, you can quickly cut high-quality slices.
3 The temperature of the freezer and the freezing head should be determined according to different tissues. If the temperature is too low, the tissue block will be too hard, the slice will be broken, and the terrace will be thin and uneven or void; on the contrary, if the temperature is too high, the hardness of the tissue block is not enough, and the slice is not easy to form or wrinkle. Data Figure 1 is for reference.
4 When slicing, if it is found that the freezing is excessive, the frozen tissue can be taken out together with the supporter, left at room temperature for a while, then sliced, or the tissue block is pressed with a thumb, or the tissue block is pressed with a thumb to soften the tissue. Slice again. In addition, raise the freezing point.
5 Slides for attaching slices should not be stored in the freezer and stored at room temperature. Because when the slice is attached, there is a temperature difference between the slide taken from the room temperature and the slice in the freezer. When the temperature of the slide is attached to the lower temperature slice, the temperature between the two substances is The difference, when they collide, the molecules move with each other to create an adsorption force that allows the slice to be firmly attached to the slide. If a refrigerated slide is used to attach the slice, the above phenomenon does not occur because the temperature is the same.
Beijing Baiou Bowei Biotechnology Co., Ltd. has Biolog microbial identification system, ultra-low temperature refrigerator, biological safety cabinet and other equipment to carry out routine molecular experiments on microbial separation and identification. The demand for life science research, biotechnology innovation and industrial development in China is actively facing the society and even collecting and collecting microbial resources. Under the premise of ensuring biosafety and protecting intellectual property rights, we provide microbial species resources, genetic resources, information resources and professional technical services for industrial and agricultural production, health, environmental protection and scientific research.
Http://
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