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CN-121977913-A - Biological tissue sample preparation sleeve liquid

CN121977913ACN 121977913 ACN121977913 ACN 121977913ACN-121977913-A

Abstract

The invention discloses a biological tissue sample preparation sleeve liquid, and belongs to the technical field of biological sample preparation. The fixing solution comprises 8-12% of aldehyde cross-linking agent, 1-3% of phosphate buffer system and the balance of purified water, wherein the dehydration solution comprises 55-70% of alcohol solvent, 10-20% of isopropanol, 10-20% of ester organic solvent, 0.5-2% of nonionic surfactant, the transparent solution comprises 50-70% of polyalcohol compound, 10-20% of ether cosolvent, 10-30% of isopropanol, the refractive index of the transparent solution is 1.47-1.52, the cleaning solution comprises 60-85% of polyalcohol solvent, 10-20% of ether solvent, 1-3% of anionic surfactant, 0.1-1% of complexing agent and the balance of purified water. Effectively reduces tissue shrinkage, keeps the cell structure intact, improves the transparentization effect, and is favorable for microscopic observation and staining analysis.

Inventors

  • LI MING
  • LI BAIZHEN
  • TANG WENPING

Assignees

  • 北京九州柏林生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260129

Claims (7)

  1. 1. A biological tissue sample preparation sleeve liquid is characterized by comprising a fixing liquid, a dehydration liquid, a transparent liquid and a cleaning liquid, wherein, The fixing solution comprises 8-12% of aldehyde cross-linking agent, 1-3% of phosphate buffer system and the balance of purified water, wherein the aldehyde cross-linking agent is formaldehyde aqueous solution, the phosphate buffer system consists of sodium dihydrogen phosphate and disodium hydrogen phosphate, the total concentration of phosphate is 0.02-0.04mol/L, and the pH value is 7.2-7.4; The dehydration liquid comprises 55-70% of alcohol solvent, 10-20% of isopropanol, 10-20% of ester organic solvent, 0.5-2% of nonionic surfactant, wherein the alcohol solvent is ethanol, the ester organic solvent is one or a combination of ethyl acetate and butyl acetate, and the nonionic surfactant is polyoxyethylene alkyl ether; the transparent liquid comprises 50-70% of a polyol compound, 10-20% of an ether cosolvent and 10-30% of isopropanol, wherein the polyol compound is selected from at least two of propylene glycol, butanediol, glycerol and triethylene glycol, the ether cosolvent is one or a combination of diethylene glycol monobutyl ether and diethylene glycol dibutyl ether, and the refractive index of the transparent liquid is 1.47-1.52; the cleaning solution comprises 60-85% of a polyol solvent, 10-20% of an ether solvent, 1-3% of an anionic surfactant, 0.1-1% of a complexing agent and the balance of purified water, wherein the polyol solvent is one or a combination of propylene glycol and glycerin, the ether solvent is diethylene glycol monobutyl ether, the anionic surfactant is sodium dodecyl sulfate, and the complexing agent is one or a combination of disodium ethylenediamine tetraacetate and sodium citrate; The alcohol solvent in the dehydration liquid and the aldehyde cross-linking agent in the fixing liquid do not generate condensation reaction under the use condition; the hydroxyl number of the polyol compound in the transparent liquid is not less than 2; the complexing agent in the cleaning fluid can carry out complexation with a cross-linked structure formed by an aldehyde cross-linking agent in the fixing fluid; the volume ratio of the fixing liquid to the dehydrating liquid to the transparent liquid to the cleaning liquid is 1:2-4:1-2:0.5-1 when the cleaning liquid is used.
  2. 2. The biological tissue sample preparation kit according to claim 1, wherein the step of preparing the fixative solution comprises: Sequentially performing active carbon adsorption treatment, cation exchange treatment, anion exchange treatment, reverse osmosis treatment and precise filtration treatment with the aperture of 0.22 mu m on raw water to obtain purified water; step two, slowly adding an aldehyde cross-linking agent into the purified water at 15-25 ℃ and mechanically stirring; step three, adding a phosphate buffer system into the mixed solution obtained in the step two and continuously stirring for 30-60 minutes; Step four, standing the obtained solution for 12-24 hours under the light-shielding condition for balancing; And fifthly, performing precise filtration on the balanced solution with the pore diameter of 0.22 mu m, and filling to obtain the fixing solution.
  3. 3. The biological tissue sample preparation kit of claim 1, wherein the step of preparing the dehydrated fluid comprises: step one, mixing ethanol and isopropanol in proportion and stirring at room temperature; step two, adding an ester organic solvent into the mixed solution obtained in the step one, and continuously stirring for 10-20 minutes; step three, adding a nonionic surfactant into the mixed solution obtained in the step two and continuously stirring for 20-30 minutes; fourthly, performing ultrasonic treatment on the obtained solution with the power of 100-200W for 5-10 minutes; step five, standing the solution subjected to ultrasonic treatment for 30-60 minutes to eliminate bubbles; and step six, performing precise filtration with the aperture of 0.22 mu m on the solution after standing, and filling to obtain the dehydration liquid.
  4. 4. The biological tissue sample preparation kit according to claim 1, wherein the step of preparing the transparent solution comprises: step one, mixing a polyol compound and an ether cosolvent in proportion and stirring for 20-40 minutes at room temperature; Step two, adding isopropanol into the mixed solution obtained in the step one and continuously stirring for 20-30 minutes; Step three, controlling the refractive index of the obtained solution within the range of 1.47-1.52 by adjusting the ratio of the polyol compound to the isopropanol; step four, standing the obtained solution for 15-30 minutes to eliminate bubbles; and fifthly, performing precise filtration with the aperture of 0.22 mu m on the solution after standing, and filling to obtain the transparent liquid.
  5. 5. The biological tissue sample preparation kit according to claim 1, wherein the cleaning solution preparation step comprises: step one, mixing a polyol solvent and an ether solvent in proportion and stirring at room temperature; step two, adding an anionic surfactant into the mixed solution obtained in the step one, and continuously stirring for 20-40 minutes; Adding a complexing agent into the mixed solution obtained in the step II, and continuously stirring until the complexing agent is completely dissolved; Step four, standing the obtained solution for 10-20 minutes to eliminate bubbles; And fifthly, performing precise filtration with the aperture of 0.22 mu m on the solution after standing, and filling to obtain the cleaning solution.
  6. 6. The biological tissue sample preparation kit according to claim 1, wherein the stability constant of the complexing agent in the washing solution is greater than the stability constant of the cross-linked structure in the fixative solution.
  7. 7. The biological tissue sample preparation kit according to claim 1, wherein the refractive index of the dehydrated liquid is less than 1.42, the refractive index of the transparent liquid is higher than the refractive index of the dehydrated liquid by more than 0.05, and the refractive index of the cleaning liquid is between the dehydrated liquid and the transparent liquid.

Description

Biological tissue sample preparation sleeve liquid Technical Field The invention relates to the technical field of biological sample preparation, in particular to a biological tissue sample preparation sleeve liquid. Background Biological tissue samples have important applications in scientific research, medical diagnosis and pathology research, and their treatment generally involves steps of fixing, dehydrating, transparentizing and washing to ensure tissue morphology stability and facilitate subsequent observation or molecular analysis. In the prior art, the liquid used for tissue fixation is usually based on aldehyde crosslinking agents, such as formaldehyde or formalin, but a single fixation liquid may cause uneven crosslinking degree under different tissue types or treatment conditions, which affects subsequent dehydration or transparent treatment. The dehydration liquid generally adopts organic solvents such as ethanol, isopropanol or acetone to gradually replace the water in the tissues. However, the refractive index, polarity and solvent selection of the conventional dehydration liquid are mostly fixed formulations, so that the conventional dehydration liquid is difficult to match with the refractive index of the subsequent transparent liquid, and the problem of discontinuous refractive index is easily generated in the stage of transparentization or optical observation. The transparent liquid mainly comprises polyalcohol or ether cosolvent and is used for improving tissue transparency and maintaining optical uniformity. The existing transparent liquid preparation depends on experience proportion, lacks a systematic refractive index control method, and also lacks chemical compatibility consideration on a cross-linked structure formed in the early stage in a tissue, so that the tissue microstructure can be uneven. The cleaning solution is used for removing residual fixative or impurities, and the prior art mostly adopts alcohols or aqueous solutions containing surfactants, but does not form a matched design with the fixative and the dehydration liquid system in chemical action, which easily results in an excessively stable cross-linked structure or is difficult to completely remove residues. In summary, most of the existing biological tissue sample processing liquids are single formulations, and lack of a systematic liquid suit design, and there are limitations in chemical and physical parameter matching among the steps of fixing, dehydrating, transparent and cleaning, and the maintenance of tissue macroscopic morphology, the continuity of liquid refractive index and the proper processing of molecular structure cannot be considered, which limits the consistency and controllability of sample preparation in histological study and molecular analysis. Disclosure of Invention The invention aims to provide a biological tissue sample preparation sleeve liquid for solving the problems of the background technology. A biological tissue sample preparation sleeve liquid comprises a fixing liquid, a dehydration liquid, a transparent liquid and a cleaning liquid, wherein, The fixing solution comprises 8-12% of aldehyde cross-linking agent, 1-3% of phosphate buffer system and the balance of purified water, wherein the aldehyde cross-linking agent is formaldehyde aqueous solution, the phosphate buffer system consists of sodium dihydrogen phosphate and disodium hydrogen phosphate, the total concentration of phosphate is 0.02-0.04mol/L, and the pH value is 7.2-7.4; The dehydration liquid comprises 55-70% of alcohol solvent, 10-20% of isopropanol, 10-20% of ester organic solvent, 0.5-2% of nonionic surfactant, wherein the alcohol solvent is ethanol, the ester organic solvent is one or a combination of ethyl acetate and butyl acetate, and the nonionic surfactant is polyoxyethylene alkyl ether; the transparent liquid comprises 50-70% of a polyol compound, 10-20% of an ether cosolvent and 10-30% of isopropanol, wherein the polyol compound is selected from at least two of propylene glycol, butanediol, glycerol and triethylene glycol, the ether cosolvent is one or a combination of diethylene glycol monobutyl ether and diethylene glycol dibutyl ether, and the refractive index of the transparent liquid is 1.47-1.52; the cleaning solution comprises 60-85% of a polyol solvent, 10-20% of an ether solvent, 1-3% of an anionic surfactant, 0.1-1% of a complexing agent and the balance of purified water, wherein the polyol solvent is one or a combination of propylene glycol and glycerin, the ether solvent is diethylene glycol monobutyl ether, the anionic surfactant is sodium dodecyl sulfate, and the complexing agent is one or a combination of disodium ethylenediamine tetraacetate and sodium citrate; The alcohol solvent in the dehydration liquid and the aldehyde cross-linking agent in the fixing liquid do not generate condensation reaction under the use condition; the hydroxyl number of the polyol compound in the transparent liquid is n