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A frozen section and brain tissue technology, applied in medical experiments and biological fields, can solve problems such as fixation, incomplete dehydration, antibody waste, and dropout, and achieve the effect of saving antibody cost, complete incubation, and complete sectioning.
Inactive Publication Date: 2018-09-25
SHANDONG UNIV OF TRADITIONAL CHINESE MEDICINE
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The above-mentioned patents are fully embedded with embedding agent. Although the fully embedded tissue can well protect the tissue shape, there are still some defects, such as: fixation and incomplete dehydration, and the direct patch method is often used after sectioning. Subsequent immunofluorescence staining, this method can cause waste of antibodies and phenomena such as stripping and falling off, and the full embedding of the embedding agent can easily lead to the formation of ice crystals
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Embodiment 1
[0034] Preparation and Immunofluorescence Staining of Rat Brain Frozen Sections
[0035] 1. Tissue sampling and fixed dehydration method
[0036] Rats were anesthetized, perfused with 4°C liquid in the left ventricle of the rat, first with normal saline until the outflow was clear, and then changed to 4% paraformaldehyde perfusion, first fast and then slow, subject to the stiffness of the whole body, and quickly removed immediately after perfusion Put the brain tissue into a 50ml centrifuge tube with 4% paraformaldehyde in advance for post-fixation, and then transfer it to 10%, 20%, and 30% sucrose solution for gradient dehydration. The sucrose solution of each gradient is generally at room temperature Under the same conditions for 4 hours to overnight, the rat brain tissue finally sank to the bottom of the tube as the standard.
[0037] 2. Freezing pretreatment method
[0038] The cryostat and the sample holder were pre-cooled in advance, and the temperature inside the micr...
Embodiment 2
[0056] Preparation and Immunofluorescence Staining of Frozen Sections of Mouse Brain Tissue
[0057] 1. Tissue sampling and fixed dehydration method
[0058] Mice were anesthetized, and the left ventricle of the mouse was perfused with 4°C liquid. First, the normal saline was perfused rapidly until the effluent was clear, and then changed to perfuse with 4% paraformaldehyde solution, first fast and then slow, subject to the stiffness of the whole body, and immediately after perfusion Take out the brain tissue, put it into a 15ml centrifuge tube with 4% paraformaldehyde solution in advance for post-fixation, and then transfer it to 10%, 20%, and 30% sucrose solution for gradient dehydration. The sucrose solution of each gradient is generally At room temperature for 4 hours to overnight, until the rat brain tissue sinks to the bottom of the tube.
[0059] 2. Freezing pretreatment method
[0060] The cryostat and the sample holder were pre-cooled in advance, and the temperature...
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Abstract
The invention discloses a method for preparing a brain tissue frozen section, belonging to the fields of biology and medical experiments. The method comprises (1) a step of tissue extraction, immobilization and dehydration, namely a step of subjecting an animal brain tissue to immobilization and gradient dehydration; (2) a step of freezing pretreatment, namely a step of washing the brain tissue with a PBS solution, wiping the brain tissue, leveling the brain tissue, wrapping the brain tissue with tin foil paper, freezing the brain tissue in a rapid freezing zone of a freezing microtome, and then taking the tissue out for subsequent treatment; (3) a step of semi-embedding treatment, namely a step of subjecting the brain tissue to semi-embedded treatment, and allowing the tissue to stand for1 minute; (4) a step of frozen sectioning, namely a step of fixing a sample holder to a probe of the microtome, and performing continuous coronal frozen sectioning; and (5) a step of section collection, namely a step of collecting sections of desired positions into a six-pore plate containing a frozen preserving fluid, and storing the sections at -20 DEG C for long time. Semi-embedding treatmentis adopted to treat the brain tissue, and therefore, influences of an embedding agent on the brain tissue are avoided, and sections are complete and free of ice crystals, and have good immunofluorescent staining properties.
Description
technical field [0001] The invention relates to a method for slicing brain tissue, in particular to a method for preparing frozen sections of brain tissue. It belongs to the fields of biology and medical experiments. Background technique [0002] Frozen section is a method of making the tissue reach a certain hardness by means of low temperature conditions and then sectioning it. It can well preserve the activity of antigens and enzymes in the tissue, so it is widely used in clinical rapid pathological diagnosis and scientific research on diseases. However, frozen section is more demanding and difficult than paraffin section, and frozen section, immunofluorescence, and immunohistochemistry have higher technical requirements than clinical pathological diagnosis. The main factors affecting the quality of frozen sections are material collection, temperature during sectioning, embedding method and storage after sectioning. Freeze appropriate tissue blocks, appropriate embeddin...
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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/42G01N1/30
CPCG01N1/30G01N1/42
Inventor 刘小菊陈丹李宁张美芝高杰王枭宇王海娟
Owner SHANDONG UNIV OF TRADITIONAL CHINESE MEDICINE
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