A dehydration reagent for biological tissues and its preparation method and application

Abstract

The present application belongs to the technical field of dehydrating agent, and particularly relates to a dehydration reagent for biological tissue as well as a preparation method and application thereof. The dehydration reagent comprises a dehydrating agent and an infiltrating agent. The dehydrating agent comprises ethanol, and the infiltrating agent comprises glycerol. Through the synergistic dehydration effect of the ethanol-glycerol mixed solution, the short board of lipid loss caused by single ethanol dehydration is made up. The lipid structure such as cell membrane and cell organ membrane is effectively protected in the dehydration process and is no longer excessively extracted, so that the full form of the cell is maintained, and the volume of the whole brain parenchyma remains stable after dehydration, effectively reducing the volume shrinkage of the biological tissue and maintaining the morphological stability.

Description

Technical Field

[0001] This invention belongs to the field of dehydrating agent technology, and specifically relates to a dehydrating agent for biological tissues, its preparation method, and its application. Background Technology

[0002] Preserving the original morphology and structure of biological tissues is fundamental to histological observation and pathological diagnosis. For delicate and fragile organs such as brain tissue, maintaining the integrity of cell morphology and overall outline during sample preparation is crucial.

[0003] Intact tissue dehydration and paraffin embedding are key pretreatment steps for preparing high-quality pathological sections. The core purpose of dehydration is to completely remove water from biological tissues so that the subsequent paraffin embedding medium can fully penetrate the biological tissues and replace the water, thereby fully supporting the tissue structure and achieving high-quality thin-layer sections.

[0004] Currently, the most widely used dehydration method in histology laboratories is gradient ethanol dehydration. The standard procedure involves immersing fixed tissue samples sequentially in ethanol solutions of increasing concentrations: 50%, 70%, 85%, 95%, and 100% ethanol, with each gradient lasting several hours, followed by paraffin embedding. However, this traditional dehydration method has significant drawbacks, leading to marked, non-uniform volume shrinkage and morphological distortion in biological tissues. This shrinkage is particularly severe in loosely structured tissues, such as mouse brain tissue, easily causing the following problems: 1) Altered tissue contours: The overall volume of the mouse brain shrinks, the surface wrinkles, and the original smooth contours are disrupted; 2) Cell morphology damage: Due to uneven shrinkage, neurons and other cells are compressed and deformed, intercellular spaces abnormally increase or collapse, disrupting the normal cell arrangement structure. These defects make it difficult to accurately observe the true anatomical structure and cell morphology of biological tissues under a microscope, reducing the reliability of research and the accuracy of diagnosis.

[0005] Therefore, providing a dehydration reagent that can effectively reduce the shrinkage of biological tissues during the dehydration process and maintain the morphological stability of biological tissues is of great significance for providing more realistic and reliable tissue samples for tissue morphology research. Summary of the Invention

[0006] The present invention aims to solve one or more technical problems existing in the prior art, and at least provide a beneficial solution. Specifically, the present invention provides a dehydrating agent that can effectively reduce the volume shrinkage of biological tissues during the dehydration process and maintain the morphological stability of biological tissues.

[0007] The inventive concept of this invention: The dehydrating agent of this invention includes a dehydrating agent and a wetting agent; the dehydrating agent includes ethanol; the wetting agent includes glycerol.

[0008] This invention uses ethanol as the main dehydrating component, leveraging its hydrophilicity and strong diffusion ability to efficiently replace and remove water from biological tissues. Glycerol molecules, with their strong hydrophilicity and large molecular size, can penetrate and enter the biological tissue while ethanol removes water, partially filling the microscopic spaces left by lost water molecules. Through the synergistic effect of ethanol and glycerol, a gentle support network is formed within the biological tissue, counteracting the internal stress caused by complete water loss and resisting tissue shrinkage during dehydration, thereby inhibiting tissue shrinkage and deformation.

[0009] Meanwhile, the present invention overcomes the shortcomings of the prior art that uses ethanol alone for dehydration, which leads to lipid loss, by using ethanol and glycerol for synergistic dehydration. The lipid structures such as cell membranes and organelle membranes are effectively protected during the dehydration process and are no longer over-extracted, thus maintaining the full shape of the cells and keeping the volume of the entire biological tissue stable after dehydration, thereby maintaining the morphological stability of the biological tissue.

[0010] Therefore, a first aspect of the present invention provides a dehydration reagent.

[0011] Specifically, the dehydrating agent includes a dehydrating agent and a wetting agent; The dehydrating agent includes ethanol; the wetting agent includes glycerin.

[0012] Preferably, in the dehydrating agent, the volume ratio of the dehydrating agent to the wetting agent is 100:(10-20); for example, the volume ratio is 100:10, 100:11, 100:12, 100:13, 100:14, 100:15, 100:16, 100:17, 100:18, 100:19, 100:20, etc.

[0013] Specifically, in the dehydration reagent of this invention, by rationally limiting the ratio of the dehydrating agent and the wetting agent, it is beneficial to effectively maintain the integrity of the cell membrane structure of biological tissues while replacing tissue water. Mouse brain tissue treated with the dehydration reagent of this invention exhibits structural features such as plump neuronal cell bodies, clearly visible nucleoli, and tightly and orderly cell arrangement in hematoxylin-eosin staining.

[0014] Preferably, the volume concentration of the ethanol is 70-100%; for example, the volume concentration is 70%, 75%, 80%, 85%, 90%, 95%, 100%, etc.

[0015] Preferably, the ethanol is an ethanol solution; more preferably, the ethanol solution includes an aqueous ethanol solution.

[0016] A second aspect of the present invention provides a method for preparing the dehydrating reagent described in the first aspect of the present invention.

[0017] Specifically, the preparation method of the dehydrating reagent includes the following steps: The dehydrating agent and the wetting agent are mixed to obtain the product.

[0018] A third aspect of the present invention provides the application of the dehydrating agent described in the first aspect of the present invention in the dehydration treatment of biological tissues.

[0019] Preferably, the application includes the following steps: The biological tissue is immersed in the dehydrating reagent for dehydration treatment.

[0020] Preferably, the biological tissue is dehydrated after fixation.

[0021] Preferably, the fixation of biological tissues is achieved by perfusing the heart with phosphate buffer and paraformaldehyde.

[0022] Preferably, the dehydration process employs a gradient dehydration method.

[0023] Preferably, the gradient dehydration treatment involves immersing the biological tissue sequentially in a dehydration reagent composed of ethanol solutions with increasing volume concentrations.

[0024] Preferably, the gradient dehydration treatment involves immersing the biological tissue sequentially in a dehydration reagent composed of 70%, 85%, and 95% ethanol solutions and glycerol; or immersing the biological tissue sequentially in a dehydration reagent composed of 75% and 100% ethanol solutions and glycerol.

[0025] Preferably, the gradient dehydration treatment involves immersing the biological tissue sequentially in a dehydration reagent composed of 70%, 85%, and 95% ethanol solutions and 10-20% glycerol solutions; or immersing the biological tissue sequentially in a dehydration reagent composed of 75% and 100% ethanol solutions and 10-20% glycerol solutions.

[0026] More preferably, the gradient dehydration treatment involves immersing the biological tissue sequentially in a dehydration reagent composed of 70%, 85%, and 95% ethanol solutions and 15% glycerol by volume; or immersing the biological tissue sequentially in a dehydration reagent composed of 75% and 100% ethanol solutions and 15% glycerol by volume.

[0027] Specifically, a glycerol volume fraction of 10-20% refers to the volume percentage of glycerol in an ethanol solution.

[0028] Preferably, the biological tissue includes intact brain tissue, and the treatment time for each gradient dehydration treatment is 2-4 hours; for example, the dehydration treatment time is 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, etc.

[0029] Preferably, when the biological tissue includes intact brain tissue, the dehydration process further includes embedding, slicing, and staining.

[0030] Preferably, the biological tissue includes brain slices, and the treatment time for each gradient dehydration treatment is 8-12 minutes; for example, the treatment time is 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, etc.

[0031] Specifically, when the biological tissue includes intact brain tissue, it needs to be fixed first, then dehydrated, and finally embedded, sectioned, and stained. When the biological tissue includes brain sections, it needs to be fixed first, then sectioned and dehydrated.

[0032] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows: (1) This invention overcomes the shortcomings of lipid loss caused by using ethanol alone for dehydration by utilizing the synergistic dehydration effect of ethanol and glycerol. Lipid structures such as cell membranes and organelle membranes are effectively protected during dehydration, preventing excessive extraction and maintaining the full shape of cells. This ensures that the volume of the entire biological tissue, such as brain parenchyma, remains stable after dehydration, effectively reducing tissue shrinkage, maintaining the overall contour of the biological tissue, and avoiding severe surface wrinkling. This provides more realistic and reliable tissue samples for tissue morphology studies. Simultaneously, it maintains the integrity of cell morphology within the brain parenchyma, ensuring tightly packed and regularly shaped neurons with normal intercellular spaces.

[0033] (2) In the dehydration reagent of the present invention, by reasonably limiting the ratio of dehydrating agent and wetting agent, a molecular protective layer is formed during the dehydration process, which effectively maintains the integrity of the cell membrane structure. Biological tissues treated with the dehydration reagent of the present invention, such as mouse brain tissue, show morphological and structural characteristics of full neuronal cell bodies, clearly visible nucleoli, and tightly and orderly arranged cells in hematoxylin-eosin staining.

[0034] (3) Paraffin sections prepared from biological tissues after dehydration treatment using the dehydration reagent of the present invention have good integrity, reducing artificial artifacts caused by tissue brittleness or deformation during the sectioning process, thus laying a solid foundation for obtaining high-quality hematoxylin-eosin staining results. Attached Figure Description

[0035] Figure 1 These are actual images of brain slices before and after dehydration treatment in the experimental group of Example 1 of this invention; Figure 2 These are actual images of brain slices before and after dehydration treatment in the control group of Example 1 of this invention; Figure 3 This is a graph showing the area ratio of brain slices before and after dehydration treatment in the experimental group and the control group in Example 1 of the present invention; Figure 4 This is a schematic diagram of the process flow for dehydration, paraffin sectioning, and hematoxylin-eosin staining of mouse whole brain samples in Example 2 of the present invention; Figure 5 These are physical images of mouse whole brain samples before and after dehydration treatment in Example 2 of the present invention; Figure 6 This is a staining image of a mouse whole brain sample treated with dehydration reagent in Example 2 of the present invention, after clearing, paraffin embedding, sectioning, and hematoxylin-eosin staining. Detailed Implementation

[0036] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0037] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0038] Example 1 This embodiment provides dehydration reagents for different gradient dehydration treatments, which consist of ethanol aqueous solutions with volume concentrations of 70%, 85%, and 95% and glycerol with a volume fraction of 15% (the volume percentage of the ethanol aqueous solution).

[0039] This embodiment also provides a method for preparing a dehydration reagent capable of gradient dehydration treatment, the specific steps of which are as follows: Ethanol aqueous solutions with volume concentrations of 70%, 85%, and 95% were mixed with glycerol with a volume fraction of 15% (the volume percentage of the ethanol aqueous solution) to obtain the desired solution.

[0040] Brain slice dehydration shrinkage experiment A brain slice dehydration and shrinkage experiment was conducted using a dehydrating reagent, and experimental and control groups were set up as follows: Experimental group: The dehydration reagents for the gradient dehydration treatment in the experimental group were 70%, 85%, and 95% volume concentration ethanol aqueous solutions, respectively, mixed with 15% volume fraction (the volume percentage of the ethanol aqueous solution) glycerol. Mice were subjected to brain fixation by first perfusing 85 mL of 0.01 M phosphate buffer into the heart, followed by perfusing 85 mL of 4% (w / v) paraformaldehyde. After heart perfusion, the mouse brain tissue was harvested, fixed by immersion in 4% (w / v) paraformaldehyde for 2 days, and then rinsed in 0.01 M phosphate buffer for 12 hours. The mouse brain was embedded in agarose and prepared into 300 μm thick brain slices using a vibratory slicer. The brain slices were then sequentially immersed in a dehydration reagent consisting of 70%, 85%, and 95% ethanol aqueous solution and 15% (by volume) glycerol in the ethanol aqueous solution for 10 min under each gradient dehydration condition.

[0041] Control group: The only difference between the control group and the experimental group is that the brain slices in the control group were successively immersed in ethanol aqueous solutions with volume concentrations of 70%, 85%, and 95% for dehydration treatment, and the dehydration treatment time under each gradient treatment condition was 10 minutes; that is, the dehydration reagent did not contain glycerol, but only ethanol, and everything else was the same as the experimental group.

[0042] Morphological observation: Images of brain slices before and after dehydration treatment in the experimental group are shown below. Figure 1 As shown in the figure, the actual brain slices of the control group before and after dehydration treatment are as follows. Figure 2 As shown. Among them, Figure 1 The left image in the image shows the actual forebrain slices from the dehydrated experimental group. Figure 1 The image on the right is a photograph of the brain slices of the experimental group after dehydration treatment; Figure 2 The left image in the image is a photograph of the brain slices from the control group after dehydration treatment. Figure 2 The image on the right is a photograph of the brain slices of the control group after dehydration treatment.

[0043] Depend on Figure 1 and Figure 2 It can be seen that brain slices treated with a dehydration reagent composed of an ethanol-water solution and glycerol maintained good morphology before and after dehydration, with smooth edges and no obvious shrinkage. In contrast, brain slices treated with only an ethanol-water solution showed severe curling and shrinkage after dehydration, with significant morphological damage. This indicates that the use of a mixture of ethanol-water solution and glycerol as a dehydration reagent in this invention can effectively reduce the volume shrinkage of biological tissues after dehydration.

[0044] Quantitative analysis: The brain slice area of ​​the experimental group and control group before and after dehydration was measured using FIJI image analysis software. The sample size was n=3, and the average value was taken. Then, the area ratio of the brain slices before and after dehydration was calculated. The area ratio results of the brain slices before and after dehydration in the experimental group and control group are shown below. Figure 3 As shown. Among them, Figure 3 The ** in the text indicates that p < 0.01.

[0045] Depend on Figure 3 As can be seen, brain slices dehydrated using the dehydrating reagent of this invention showed a reduction in area of ​​approximately 5% after dehydration, remaining at 95% of their original size, indicating a morphological retention rate of up to 95%. In contrast, brain slices in the control group, dehydrated only with ethanol, showed a sharp reduction in area of ​​30% after dehydration, remaining at 70% of their original size, with a morphological retention rate of only 70%. This quantitative data fully demonstrates that the dehydrating reagent of this invention can effectively resist tissue shrinkage caused by the dehydration process, reduce the volume shrinkage of biological tissues, and maintain the morphology of biological tissues.

[0046] The above demonstrates that the dehydration reagent of the present invention can effectively reduce the volume shrinkage of biological tissue samples during the dehydration process through the synergistic effect of ethanol and glycerol.

[0047] Example 2 Paraffin sections of mouse brain and hematoxylin-eosin staining Dehydration and embedding: Mouse brains were fixed by perfusing 85 mL of 0.01 M phosphate buffer into the heart and then perfusing 85 mL of 4% (w / v) paraformaldehyde. After heart perfusion, the mouse brain tissue was removed and soaked in 4% (w / v) paraformaldehyde for 2 days, then soaked and rinsed in 0.01 M phosphate buffer for 12 hours. Then, the tissue was successively placed in a dehydration reagent consisting of 75% and 100% ethanol aqueous solution and 15% (volume percentage of ethanol aqueous solution) glycerol for 2 hours each. After that, the tissue was cleared with xylene for 4 hours, immersed in paraffin at 60℃ for 12 hours, embedded in paraffin, sectioned, and sectioned to a thickness of 5 μm. Finally, the tissue was stained with hematoxylin and eosin.

[0048] The hematoxylin-eosin staining process is as follows: xylene dewaxing for 25 min, xylene dewaxing for 25 min, 100% alcohol for 5 min, 100% alcohol for 5 min, 95% alcohol for 5 min, 80% alcohol for 5 min, distilled water for 1 min, hematoxylin staining solution for 12 min, tap water washing for 1 min, 1% (v / v) hydrochloric acid alcohol (hematoxylin differentiation solution) for 15 s, running water rinsing for 20 min, eosin staining solution for 12 min, 80% alcohol for 15 s, 95% alcohol for 5 min, 100% alcohol for 10 min, 100% alcohol for 10 min, xylene clearing for 12 min, xylene clearing for 12 min, then mounting with neutral resin and microscopic examination.

[0049] The schematic diagram of the process for dehydration, paraffin sectioning, and hematoxylin-eosin (HE) staining of mouse whole brain samples in Example 2 is shown below. Figure 4 As shown.

[0050] Images of mouse whole brain samples before and after dehydration treatment are shown below. Figure 5 As shown. Among them, Figure 5The left image in the image shows a physical photograph of a mouse whole brain sample before dehydration treatment. Figure 5 The image on the right is a photograph of a whole brain sample from a mouse after dehydration.

[0051] The staining images of mouse whole brain samples treated with the dehydration reagent of this invention after clearing, paraffin embedding, sectioning, and hematoxylin-eosin staining are shown below. Figure 6 As shown. Among them, Figure 6 The left image in the image shows the staining pattern after hematoxylin-eosin staining. Figure 6 The right image in the image is a magnified view of the area within the box in the left image.

[0052] Depend on Figure 5 and Figure 6 As can be seen, the mouse brain tissue treated with the dehydration reagent of this invention maintained its morphology well and exhibited good morphological preservation in hematoxylin-eosin staining. Neuronal cell bodies in the cortical region were plump and morphologically intact; the nuclei and nucleoli were clearly visible after hematoxylin staining, and the cytoplasm stained uniformly with eosin. Neuronal cells were arranged tightly and orderly, with distinct hierarchical structures, and the intercellular spaces maintained a normal physiological state. These results indicate that the ethanol-glycerol gradient dehydration system used in this invention can effectively maintain the microstructural integrity of mouse brain tissue, providing a high-quality sample basis for subsequent histological analysis.

[0053] In summary, this invention overcomes the lipid loss problem caused by using ethanol alone as a dehydrating agent through the synergistic dehydration effect of ethanol and glycerol. Lipid structures such as cell membranes and organelle membranes are effectively protected during dehydration, preventing excessive extraction and maintaining the plump shape of cells. This ensures the overall volume of the brain parenchyma remains stable after dehydration, effectively reducing tissue shrinkage, maintaining the overall contour of the tissue, and avoiding severe surface wrinkling. This provides more realistic and reliable tissue samples for histological studies. Simultaneously, it preserves the integrity of cell morphology within the brain parenchyma, maintaining tightly packed and regularly shaped neurons with normal intercellular spaces.

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A dehydration reagent, characterized in that, The dehydrating agent includes a dehydrating agent and a wetting agent; The dehydrating agent includes ethanol; the wetting agent includes glycerin.

2. The dehydrating reagent according to claim 1, characterized in that, In the dehydrating agent, the volume ratio of the dehydrating agent to the wetting agent is 100:(10-20).

3. The dehydrating reagent according to claim 1, characterized in that, The volume concentration of the ethanol is 70-100%.

4. The method for preparing the dehydrating reagent according to any one of claims 1-3, characterized in that, The preparation method includes the following steps: The dehydrating agent and the wetting agent are mixed to obtain the product.

5. The use of the dehydrating agent according to any one of claims 1-3 in the dehydration treatment of biological tissues.

6. The application according to claim 5, characterized in that, The application includes the following steps: The biological tissue is immersed in the dehydrating reagent for dehydration treatment.

7. The application according to claim 6, characterized in that, The dehydration treatment employs a gradient dehydration method; and / or, the biological tissue undergoes dehydration treatment after fixation.

8. The application according to claim 7, characterized in that, The gradient dehydration treatment involves immersing biological tissues sequentially in a dehydration reagent composed of ethanol with increasing volume concentrations.

9. The application according to claim 8, characterized in that, The biological tissue includes intact brain tissue, and the treatment time for each gradient dehydration treatment is 2-4 hours.

10. The application according to claim 8, characterized in that, The biological tissue includes brain slices, and during the gradient dehydration treatment, the treatment time for each gradient is 8-12 minutes.

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