CN115615783A - Preparation method of paraffin tissue section - Google Patents
Preparation method of paraffin tissue section Download PDFInfo
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- G—PHYSICS
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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Abstract
The application relates to a preparation method of a paraffin tissue section, which comprises the following steps: pre-cooling the tissue wax block; wetting the surface to be cut of the precooled tissue wax block with water; cutting the wetted tissue wax block to form a section with a wetted surface; placing the slices on the water surface in a manner that the wet surfaces are directly contacted with water to spread the slices; sequentially baking, dewaxing and hydrating the slices after the slicing is finished, and preparing hydrated slices; treating the hydrated slices in a sodium citrate antigen repairing solution at 50-90 ℃ for 2-3 min to prepare slices treated by the sodium citrate antigen repairing solution; cooling the slices treated by the sodium citrate antigen retrieval solution, and then washing the slices with water to prepare washed slices; and staining the cleaned section to prepare a paraffin tissue section. The preparation method can effectively avoid tissue cracking, and the prepared paraffin tissue section has a complete cell structure and bright and clear dyeing.
Description
Technical Field
The application relates to the technical field of biological histology, in particular to a preparation method of a paraffin tissue section.
Background
Paraffin sectioning is the most widely used method in conventional histological sectioning techniques. The paraffin section is not only used for observing the morphological structure of normal cell tissues, but also is an indispensable component in the research of pathological morphology, biological morphology, qualitative analysis of cell structure and change.
The conventional paraffin tissue section method comprises the steps of material taking, fixing, washing and dehydrating, transparentizing, wax dipping, embedding, section and patch pasting, dewaxing, dyeing, dehydrating, transparentizing, mounting and the like. The conventional method has high requirements on the quality of wax blocks, the quality of blades and the proficiency of personnel, is easy to cause tissue lobe and indistinct in dyeing, and is not beneficial to the observation and judgment of parenchymal organs such as ovaries, testicles, pancreas and the like.
Disclosure of Invention
Based on the method, the preparation method of the paraffin tissue section can effectively avoid tissue cracking, and the prepared paraffin tissue section is complete in cell structure, uniform and clear in dyeing.
The technical scheme for solving the technical problems is as follows:
a preparation method of paraffin tissue slices comprises the following steps:
pre-cooling the tissue wax block;
wetting the surface to be cut of the precooled tissue wax block with water;
cutting the moistened tissue wax block to form a section with a moist surface;
placing the slices on the water surface in a manner that the wet surface is directly contacted with water so as to carry out the spreading;
sequentially baking, dewaxing and hydrating the slices after the slicing is finished, and preparing hydrated slices;
heating the hydrated slices in a sodium citrate antigen repairing solution at 50-90 ℃ for 2-3 min to prepare slices treated by the sodium citrate antigen repairing solution;
cooling the slices treated by the sodium citrate antigen retrieval solution, and then washing the slices with water to prepare washed slices; and
and dyeing the cleaned section to prepare a paraffin tissue section.
The application provides a preparation method of paraffin tissue slices, which reduces static electricity generated during continuous slicing of tissues by wetting a paraffin block section with water in a slicing process and avoids curling and cracking of the tissues by using the tension of the water; in addition, the method adds the sodium citrate antigen repairing solution for heat treatment before the tissue section dyeing step, has good pH regulation and buffering performance, and can effectively reduce the cracking condition of the section after tissue dyeing. Therefore, the prepared conventional paraffin tissue section has a complete cell structure, is uniformly and clearly dyed, and is convenient for observing the morphological change of the tissue cells.
In some embodiments, the step of preparing the section treated with the sodium citrate antigen retrieval solution further comprises: heating the sodium citrate antigen retrieval solution to boiling.
In some embodiments, the pre-cooling temperature is 0-4 ℃, and the pre-cooling time is 20-30 min.
In some embodiments, the operation of wetting the to-be-cut surface of the pre-cooled tissue wax block with water comprises:
and wetting the section to be cut of the tissue wax block by using water-wetted dust-free paper towels or mirror wiping paper.
In some embodiments, the sections are stained by hematoxylin-eosin staining method, wherein the staining time of hematoxylin staining solution is 20-30 s.
In some embodiments, the step of staining the section with hematoxylin-eosin staining comprises:
placing the cleaned section in hematoxylin staining solution for staining for 20-30 s, and preparing a hematoxylin stained section;
rinsing the section stained by the hematoxylin, soaking the section in hydrochloric acid alcohol with the volume fraction of 0.1% for differentiation for 3-5 s, and preparing a differentiated section;
placing the differentiated slices in water to turn blue, and preparing the slices which turn blue; and
and placing the slices subjected to blue returning into absolute ethyl alcohol, then carrying out eosin dyeing treatment, and rinsing after staining in an eosin dyeing solution to obtain the hematoxylin-eosin dyed slices.
In some of these embodiments, the preparation method further comprises the steps of sequentially fixing, dehydrating, clearing, waxing and embedding the tissue sample to prepare a tissue wax block.
In some of these embodiments, the operation of fixing the tissue sample comprises: soaking the tissue sample in a fixing solution for fixing for 24-48 h; and
and (4) washing the fixed tissue sample, and removing the fixing liquid.
In some of these embodiments, the dehydrating comprises:
soaking the fixed tissue samples in the following solutions in sequence from low ethanol concentration to high ethanol concentration: 30% ethanol solution, 50% ethanol solution, 75% ethanol solution, 80% ethanol solution, 95% ethanol solution and 95% ethanol solution, wherein the soaking time of each ethanol solution is 1-2 h independently; and
soaking the tissue sample treated by the ethanol solution with the volume fraction of 95% for 2 times by using absolute ethanol, wherein the soaking time is 45-60 min each time.
In some of these embodiments, the transparent operation comprises:
and sequentially soaking the dehydrated tissue sample in a solution containing a clearing agent according to the sequence of the concentration of the clearing agent from low to high, wherein the clearing agent comprises one or more of dimethylbenzene, benzene, chloroform and n-butyl alcohol.
Drawings
FIG. 1 is a microscopic view showing the staining of paraffin tissue sections of examples 1 to 5 and comparative examples 1 to 10;
FIG. 2 is a microscopic view of the stained paraffin tissue sections of examples 6 to 21;
FIG. 3 is a microscopic view of the stained paraffin tissue sections of examples 22 to 37;
FIG. 4 is a microscopic examination image of paraffin tissue section staining of examples 38 to 53 。
Detailed Description
In order to make the aforementioned objects, features and advantages of the present application more comprehensible, preferred embodiments of the present application are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. This application is capable of embodiments in many different forms than those described herein and that modifications may be made by one skilled in the art without departing from the spirit and scope of the application and it is therefore not intended to be limited to the specific embodiments disclosed below.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the present application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. The reagents or instruments used herein are not indicated by manufacturers, and are all conventional products that can be obtained by purchase.
In the conventional pathological section making process, factors causing tissue cracking, blurred staining and unclear cell structure have many aspects including any aspects in the processes of material taking, fixing, dehydration, transparency, waxing, embedding, slicing, slice unfolding, slice baking, dewaxing, hydration, staining and the like.
Based on this, the technical personnel of this application break the prior art and adopt water wetting tissue tangent plane creatively, form the tissue that has the wetting face, carry out follow-up section operation. The technicians of the application further perform a great deal of research on obtaining the section with complete cell structure and clear staining of the tissue section, and find out by chance: after the tissue section is subjected to heat treatment in the sodium citrate antigen retrieval solution, conventional tissue dyeing is performed, so that the cracking condition of the section after tissue dyeing can be effectively reduced, and the prepared tissue section has a complete cell structure and is uniformly and clearly dyed.
Accordingly, an embodiment of the present invention provides a method for preparing a paraffin tissue section, the method comprising steps (1) to (8):
material taking and fixing in the step (1): taking a fresh tissue sample, removing unrelated tissues, and then putting the tissue sample into a fixing solution for fixing.
Dehydrating and transparent in step (2): and (2) sequentially dehydrating the tissue samples fixed in the step (1) by using an ethanol solution with a gradient from low concentration to high concentration as a dehydrating agent, and sequentially replacing the dehydrating agent in the tissue by using a clearing agent with a gradient from low concentration to high concentration.
And (3) wax dipping and embedding: and (3) sequentially soaking the tissues in the step (2) in three-cylinder paraffin solution for waxing treatment, and adding paraffin into the waxed tissues for embedding.
Slicing in step (4): pre-cooling the tissue wax block; wetting the surface to be cut of the precooled tissue wax block by water; the wetted tissue wax mass is cut to form a section with a wetted surface.
And (5) unfolding and baking slices: placing the slices in the step (4) on the water surface in a manner that the wet surfaces are directly contacted with water so as to spread the slices; and then placing the tissue section on a staining rack and baking the tissue section in an oven.
Dewaxing and hydrating in the step (6): and (4) putting the slices in the step (5) into a three-cylinder xylene solution in sequence for dewaxing treatment, and then soaking the tissue slices in ethanol solution with gradient from high concentration to low concentration and two cylinders of ultrapure water for hydration treatment in sequence.
Step (7), dyeing: treating the hydrated slices in a sodium citrate antigen repairing solution at 50-90 ℃ for 2-3 min to prepare slices treated by the sodium citrate antigen repairing solution; cooling the slices treated by the sodium citrate antigen retrieval solution, and then washing the slices with water to prepare washed slices; and staining the washed section.
And (8) dehydrating and transparentizing: and (5) sequentially soaking the tissue slices in the step (7) in three cylinders of absolute ethyl alcohol solution, wherein the time of dehydration treatment is 1min each time, and then sequentially soaking the tissue slices in two cylinders of xylene tissue clearing agent solution, wherein the time of clearing treatment is 2min each time.
The preparation method of the paraffin tissue section comprises the steps of material taking, fixing, dehydration, transparency, waxing, embedding, section cutting, section spreading, section baking, dewaxing, hydration, dyeing and the like. On one hand, in the slicing process, the wax block section is wetted by water, so that the static electricity generated in the continuous slicing of the tissue is reduced, and the tissue is prevented from curling and cracking by using the tension of the water; on the other hand, improper steps such as tissue fixing, embedding and the like can cause protein degeneration and can cause dye rejection of tissues, sodium citrate antigen repairing liquid is added for heat treatment before the step of tissue section dyeing, the sodium citrate antigen repairing liquid is commonly used for immunohistochemical antigen repairing, and the fixing effect of the fixing liquid on the tissues is changed; in addition, the method has good PH regulation effect, can reduce the PH value of the surface of the section, and improves the staining effect of hematoxylin on cell nucleus. Therefore, the prepared conventional paraffin tissue section has a complete cell structure, is uniformly and clearly dyed, and is convenient for observing the morphological change of the tissue cells.
In some embodiments, the fresh tissue sample of step (1) includes, but is not limited to, mouse spleen, liver, pancreas, lung, kidney, testis, ovary tissue.
Further, the tissue sample may be selected according to an actually desired observation target.
In some embodiments, the fixing solution in step (1) includes at least one of paraformaldehyde with a volume fraction of 4%, formalin with a volume fraction of 10%, neutral formalin, and formalin-ethanol-acetic acid mixed fixing solution.
In some examples, the fixation time of the fixation solution in the step (1) is 24-48 h. It will be appreciated that the fixed time may be adjusted according to the tissue mass size.
In some embodiments, step (1) further comprises the step of washing the fixed tissue sample to remove the fixative. Optionally, after fixation is completed, the tissue is rinsed in running water for 20min to 30min, it being understood that the rinsing removes the residual fixative in the tissue.
In some embodiments, the dehydration treatment in step (2) comprises sequentially soaking the tissue sample in the following solutions in the order of ethanol concentration from low to high: 30% by volume of ethanol solution, 50% by volume of ethanol solution, 75% by volume of ethanol solution, 80% by volume of ethanol solution, 95% by volume of ethanol solution and 95% by volume of ethanol solution, wherein the soaking time of each ethanol solution is independently 1-2 h; and soaking the tissue sample treated by the ethanol solution with the volume fraction of 95% for 2 times by adopting absolute ethyl alcohol, wherein the soaking time is 45-60 min each time. It is understood that in other embodiments, the selection of the concentration of ethanol during the dehydration process is not limited to the above.
In some embodiments, the operation of transparency in step (2) comprises: and sequentially soaking the dehydrated tissue sample in a solution containing a clearing agent according to the sequence of the concentration of the clearing agent from low to high, wherein the clearing agent comprises one or more of dimethylbenzene, benzene, chloroform and n-butyl alcohol.
In some specific examples, the clearing agent in step (2) is xylene, wherein the xylene from low to high concentration gradients is a mixture of xylene in a volume ratio of 1:1, the mixed solvent of absolute ethyl alcohol and dimethylbenzene, dimethylbenzene I and dimethylbenzene II, and the time of each transparent treatment is 10-20 min. Wherein, I and II in the dimethylbenzene are the same substances with different numbers.
It can be understood that the transparent time can be adjusted according to the size of the tissue block, and the tissue can be observed to be bright under illumination.
In some embodiments, the temperature of the paraffin solution during the wax dipping treatment in the step (3) is 58-60 ℃, and the wax dipping time per cylinder is 30-60 min.
In some of these embodiments, the embedding in step (3) comprises: placing the tissues after being waxed in an embedding groove, adding paraffin, placing the tissues in the center of a bottom groove, covering an embedding box with a mark, pouring the paraffin, and cooling and solidifying; after solidification, the tissue wax block is frozen at minus 20 ℃ for 10min to 20min, the embedding groove is taken down, and the tissue wax block is stored at normal temperature or 4 ℃.
In some embodiments, the pre-cooling temperature in the step (4) is 0-4 ℃, and the pre-cooling time is 20-30 min.
In some specific examples, the operation of wetting the to-be-cut surface of the pre-cooled tissue wax block in the step (4) comprises:
the section to be cut of the tissue wax block is moistened by water-moist dust-free paper towels or mirror wiping paper so as to form water stains on the section to be cut. It is understood that in other embodiments, the method for wetting the surface to be cut of the tissue wax block is not limited to the above, and is not limited to the use of a non-woven fabric or a dust-free paper towel, but can be other methods as long as the surface to be cut can be wetted without damaging and contaminating the surface to be cut, for example, spraying water to the surface to be cut.
When continuous slicing is carried out, the section to be sliced needs to be wetted once every time.
Through wetting the surface to be cut of the tissue block, the static electricity generated during continuous slicing of the tissue can be reduced, and the tissue is prevented from curling and cracking by using the tension of water.
In some specific examples, in the step (5), the section with the water stain in the step (4) is downwards faced by tweezers, placed in clean water for flattening, then the slices are fished out, and transferred to a clean water bath for the slice spreading treatment.
Optionally, the temperature of the water bath for the slide treatment in the step (5) is 48-50 ℃, and the slide treatment time is 5-15 s.
In some embodiments, the temperature of the baking treatment in the step (5) is 58-60 ℃, and the time is 1-2 h.
In some of the embodiments, the dewaxing treatment time in step (6) is 10min to 15min.
In some embodiments, the ethanol solution with high concentration to low concentration gradient in the step (6) is absolute ethanol I, absolute ethanol II, ethanol with volume fraction of 95%, ethanol with volume fraction of 80% and ethanol with volume fraction of 75%, and the hydration treatment time is 3min to 5min each time. It is to be understood that the content of ethanol in the ethanol solution in the hydration treatment is not limited to the above.
In some alternative specific examples, the temperature of the sodium citrate antigen retrieval solution of step (7) is 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃ or 90 ℃; the treatment time of the sodium citrate antigen retrieval liquid is 2min, 2.5min and 3min.
In some embodiments, the step (7) of preparing the slice heat-treated with the sodium citrate antigen retrieval solution further comprises: heating the sodium citrate antigen retrieval solution to boiling.
In some embodiments, the sodium citrate antigen retrieval solution used in step (7) is obtained by diluting 100 × sodium citrate antigen retrieval solution to 1 × sodium citrate antigen retrieval solution.
In some embodiments, the section is stained in the step (7) by adopting a hematoxylin-eosin staining method, wherein the staining time of the hematoxylin staining solution is 20-30 s.
Optionally, placing the cleaned section in hematoxylin staining solution for staining for 20-30 s, and preparing a hematoxylin stained section; rinsing the section stained by hematoxylin, soaking the section in hydrochloric acid alcohol with the volume fraction of 0.1% for differentiation for 3-5 s, and preparing a differentiated section; putting the differentiated slices into water to turn blue, and preparing the blue-turned slices; and placing the rewet section in absolute ethyl alcohol for 30s, then carrying out eosin dyeing treatment, and rinsing in absolute ethyl alcohol after dyeing in an eosin dyeing solution to obtain the hematoxylin-eosin dyed section.
In some embodiments, step (8) is followed by step (9) of mounting. Specifically, the step of mounting includes: and (5) dropwise adding neutral gum into the center of the glass slide in the step (8), covering a cover glass, and placing the glass slide in a ventilated place for airing to obtain a permanent section of the tissue.
It will be appreciated that in other embodiments, steps (1) - (3) may be omitted, in which case commercial tissue wax blocks may be purchased directly.
One embodiment of the present application provides a tissue section, which is prepared by the above method for preparing a paraffin tissue section.
The preparation method can be used for other dyeing, such as immunohistochemistry, masson dyeing and the like, is used as an optimization pretreatment step before dyeing, is beneficial to improving the slice quality and dyeing effect, effectively avoids tissue cracking, enables the focus part under a microscope to be clearer in pathological diagnosis, and further improves the diagnosis accuracy.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
While the present application will be described in conjunction with specific embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
Example 1
(1) Material taking and fixing: taking a fresh mouse pancreas tissue sample, removing irrelevant tissues, fixing in 4% paraformaldehyde solution for 24h, and flushing the tissues for 30min by running water after the fixation is finished.
(2) Dehydrating and transparent: sequentially soaking the tissues in 30%, 50%, 75%, 80%, 95% and 95% ethanol solutions in volume fraction for 2h each time; soaking the tissue in 100% anhydrous ethanol for 2 times, each time for 45min, and dehydrating; the tissue was then sequentially soaked in absolute ethanol + xylene (v: v = 1), xylene i, xylene ii for 10min each time.
(3) Wax dipping and embedding: melting paraffin in advance, and sequentially soaking tissues in three paraffin solutions I, II and III at 60 ℃ for 30min,1h and 1h respectively; and then placing the tissues after being waxed in a metal embedding groove, adding paraffin, placing the tissues in the center of the bottom groove by using forceps, covering the marked embedding box, pouring the paraffin in the embedding box, and moving the embedding box to a freezing table for solidification. Freezing at-20 deg.C for 20min after solidification, taking down the embedding groove, and storing at normal temperature.
(4) Slicing: pre-cooling the tissue wax block for 20min at 4 ℃, dipping a proper amount of room-temperature clean water by using a dust-free paper towel, wetting the pre-cooled surface to be cut by using one corner of the paper towel, and cutting the wetted tissue wax block to form a section with a wetted surface.
(5) Exhibition of slices: transferring the tissue slices by using tweezers, placing the slices with water stains on a clear water surface of a slice spreading machine, fishing out the slices by using a glass slide after the slices are slightly flattened, transferring the slices to the slice spreading machine in a clear water bath at 50 ℃, spreading the slices after the tissue slices are flattened without folds, fishing out tissues, adhering the tissues to the glass slide, obliquely placing the slices at 37 ℃ at a temperature of 45 ℃, and draining water.
(6) Baking and dewaxing: placing the tissue slices on a staining rack, baking the tissue slices for 2h in an oven at 60 ℃, and then placing the tissue slices in dimethylbenzenes I, II and III for dewaxing treatment, wherein the treatment time is 10min respectively.
(7) Hydration: sequentially soaking the tissue in absolute ethyl alcohol I, absolute ethyl alcohol II, 95% ethanol solution, 80% ethanol solution, 75% ethanol solution and ultrapure water I and II for 5min.
(8) Dyeing: heating 1 x sodium citrate antigen retrieval liquid manufacturer 1 (purchased from biological engineering limited company, model number E673001-0100) for 5min to boil, placing the hydrated tissue slice in sodium citrate antigen retrieval liquid, heating at 90 ℃ for 2min, naturally cooling, washing with ultrapure water for 3 times, 5min each time, preparing the cleaned slice, and dyeing the cleaned slice, wherein the dyeing operation comprises the following steps: placing the cleaned section in hematoxylin staining solution for staining for 25s, and rinsing in tap water for 1min;
soaking the rinsed slices in 0.1% hydrochloric acid alcohol by volume fraction for differentiation for 5s;
placing the differentiated slices in tap water, and turning blue for 10min;
and (3) placing the slices subjected to blue returning in absolute ethyl alcohol for 30s, then carrying out eosin dyeing treatment, dyeing in an eosin dyeing solution for 5s, and rinsing in absolute ethyl alcohol for 30s.
(9) Dehydrating and transparent: soaking the tissue slices in anhydrous ethanol solutions I, II and III for 1min respectively; then, the tissue slices were soaked in the tissue clearing agent solutions I and II for 2min, respectively.
(10) Sealing: and (4) dropwise adding a proper amount of neutral gum, covering a cover slip, and airing in a ventilated place.
The obtained paraffin tissue section was observed under an optical microscope, and the obtained tissue image was shown in FIG. 1-A. As shown in FIG. 1-A, the mouse pancreas tissue has no cracks, no obvious cracks and oversized intercellular spaces, and the staining is clear and vivid.
Examples 2 to 5
Examples 2 to 5 the method of preparing paraffin tissue sections was substantially the same as in example 1, except that the tissue samples were different, the specific test conditions and the corresponding effects were as shown in fig. 1, and the obtained tissue sections were observed under an optical microscope, and the results were as shown in fig. 1B to E, and the tissue sections were intact as shown in fig. 1B to E, and the mouse tissue structure was free from cracks, breaks and cavities, and was clearly and vividly stained.
Examples 6 to 21
Examples 6 to 21 the preparation of paraffin tissue section method and example 1 basically the same, the difference is the staining step is different, sodium citrate antigen repair liquid manufacturer 2 for mai new biotechnology development limited company, model MVS-0101, the specific test conditions as shown in figure 2, will make tissue section under optical microscope observation, the results as shown in figure 2A-P, in figure 2A/E/I/M and B/F/J/N are respectively water and PBS heat treatment to get the section, can be seen, the organization structure appears a plurality of crack phenomenon, the crazing is more, the staining is unclear; as shown in O-P in FIG. 2, even when the sodium citrate antigen retrieval solution is used at normal temperature (25 ℃), the tissue cracking cannot be alleviated; the slices prepared by heating the sodium citrate antigen repairing solution of different manufacturers at 55 ℃, 65 ℃ and 85 ℃ have complete tissue slices as shown in C-D, G-H and K-L in figure 2, the pancreas tissues of mice have no cracks, no cracks or overlarge cell gaps, and the staining is clear and bright.
Examples 22 to 37
Examples 22 to 37 the method of preparing paraffin tissue sections was substantially the same as in example 1, except that the staining procedure was different, the sodium citrate antigen retrieval solution manufacturer 2 was a mai new biotechnology development limited company, model number MVS-0101, the specific test conditions are shown in fig. 3, the prepared tissue sections were observed under an optical microscope, the results are shown as a to P in fig. 3, a/E/I/M and B/F/J/N in the figure are sections obtained by heating water and PBS, respectively, it can be seen that the tissue structure exhibited a plurality of cracks, more cracks, fractures and holes, and staining was not distinct; as shown in O to P in FIG. 3, even when the sodium citrate antigen retrieval solution is used at normal temperature (25 ℃), the tissue cracking cannot be alleviated; the tissue sections of the sodium citrate antigen repairing solution prepared by different manufacturers through heating treatment at 55 ℃, 65 ℃ and 85 ℃ are complete as shown in C-D, G-H and K-L in figure 3, the tissue tissues of the ovary part of the mouse have no cracks, no obvious cracks or overlarge cell gaps, and the staining is clear and bright.
Examples 38 to 53
Examples 38 to 53 methods for preparing paraffin tissue sections were substantially the same as in example 1, except that the tissues were different and the staining procedure was different, the sodium citrate antigen retrieval solution manufacturer 2 was a maiden biotechnology development limited company, model MVS-0101, the specific test conditions are shown in fig. 4, the prepared tissue sections were observed under an optical microscope, the results are shown as a to P in fig. 4, a/E/I/M and B/F/J/N in fig. 4 were sections obtained by heating water and PBS, respectively, it was found that mouse testicular tissue was significantly cracked, had many cracks, had too large intercellular spaces, and was poorly stained; as shown in O to P in FIG. 4, even when the sodium citrate antigen retrieval solution was used at normal temperature (25 ℃), the tissue cracking could not be alleviated; the section prepared by heating the sodium citrate antigen repairing solution of different manufacturers at 55 ℃, 65 ℃ and 85 ℃ has few tissue cracks, no obvious cracks and overlarge cell gaps, and clear and bright dyeing as shown in C-D, G-H and K-L in figure 4.
Comparative example 1
Comparative example 1 a paraffin tissue section was prepared in substantially the same manner as in example 1, except that comparative example 1 was not optimized for staining. Specifically, the method comprises the following specific steps of treating a tissue sample to obtain a hydrated tissue section, and staining the hydrated tissue section:
(9) Dyeing: placing the hydrated section in hematoxylin staining solution for staining for 1min, and rinsing in tap water for 1min;
soaking the rinsed slices in hydrochloric acid alcohol with the volume fraction of 0.1% for differentiation for 5s;
placing the differentiated slices in tap water, and returning blue for 10min;
and (3) placing the slices after returning blue in absolute ethyl alcohol for 30s, then carrying out eosin dyeing treatment, dyeing in an eosin dyeing solution for 5s, and rinsing in absolute ethyl alcohol for 30s.
The obtained paraffin tissue section is observed under an optical microscope, and the obtained tissue picture is shown as figure 1-F, and the mouse pancreas tissue in the figure 1-F is obviously cracked, has more cracks and is indistinct in dyeing.
Comparative examples 2 to 5
Comparative examples 2 to 5 the method for preparing paraffin tissue sections was substantially the same as in comparative example 1, except that the tissue samples were different, and the prepared tissue sections were observed under an optical microscope, and the results are shown in fig. 1, G to J, which are steps of heating treatment without using a dyed sodium citrate antigen-repairing solution in the dyeing process, and it was found that the mouse tissues had cracked, many cracks, and unclear dyeing.
Comparative example 6
Comparative example 6 a paraffin tissue section was prepared in substantially the same manner as in example 1, except that comparative example 6 was not optimized for sectioning nor for staining, and the specific steps were as follows:
(4) Slicing: the embedded wax block is directly sliced.
(9) Dyeing: placing the hydrated section in hematoxylin staining solution to stain for 1min, and rinsing in tap water for 1min;
soaking the rinsed slices in 0.1% hydrochloric acid alcohol by volume fraction for differentiation for 5s;
placing the differentiated slices in tap water, and returning blue for 10min;
and (3) placing the slices after returning blue in absolute ethyl alcohol for 30s, then carrying out eosin dyeing treatment, dyeing in an eosin dyeing solution for 5s, and rinsing in absolute ethyl alcohol for 30s.
The obtained paraffin tissue section is observed under an optical microscope, and the obtained tissue picture is shown as figure 1-K, wherein the tissue of the pancreas part of a mouse in the figure 1-K is folded, cracked and cracked, and the staining is not bright.
Comparative examples 7 to 10
Comparative examples 7 to 10 the method for preparing paraffin tissue sections was substantially the same as in comparative example 6, except that the tissue samples were different, and the prepared tissue sections were observed under an optical microscope, and the results were as shown by L to O in fig. 1, wherein L to O in fig. 1 indicate that the sections were not subjected to the wetting treatment, and the heating treatment step was not performed with the dyed sodium citrate antigen retrieval solution, and it was found that the tissues of the mouse were partially folded, cracked, too large in cell gaps, and indistinct in dyeing.
The results of the comprehensive graphical representation show that: compared with the preparation method of the tissue section of a comparative example, the tissue section prepared by the preparation method of the paraffin tissue section has a clear and complete structure, is dyed vividly and has no tissue crack.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several implementation modes of the present application, and the description thereof is specific and detailed, but not construed as limiting the scope of the claims. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, and these are all within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. The preparation method of the paraffin tissue section is characterized by comprising the following steps:
pre-cooling the tissue wax block;
wetting the surface to be cut of the precooled tissue wax block with water;
cutting the moistened tissue wax block to form a section with a moist surface;
placing the slices on a water surface in a manner that the wet surface is directly contacted with water so as to carry out the spreading;
baking, dewaxing and hydrating the sliced slices after the slicing is finished in sequence to prepare hydrated slices;
heating the hydrated slices in a sodium citrate antigen repairing solution at 50-90 ℃ for 2-3 min to prepare slices treated by the sodium citrate antigen repairing solution;
cooling the slices treated by the sodium citrate antigen retrieval solution, and then washing the slices with water to prepare washed slices; and
and dyeing the cleaned section to prepare a paraffin tissue section.
2. The method for preparing a cell according to claim 1, further comprising, before the step of preparing the slice treated with the sodium citrate antigen retrieval solution: heating the sodium citrate antigen retrieval solution to boiling.
3. The preparation method according to claim 1, wherein the pre-cooling temperature is 0-4 ℃, and the pre-cooling time is 20-30 min.
4. The preparation method according to any one of claims 1 to 3, wherein the operation of wetting the to-be-cut surface of the pre-cooled tissue wax block with water comprises:
and wetting the section to be cut of the tissue wax block by using water-wetted dust-free paper towels or lens wiping paper.
5. The method according to claim 1, wherein the sections are stained by a hematoxylin-eosin staining method, wherein the staining time of the hematoxylin staining solution is 20 to 30 seconds.
6. The method of claim 5, wherein the step of staining the section using hematoxylin-eosin staining comprises:
placing the cleaned section in hematoxylin staining solution for staining for 20-30 s to prepare a hematoxylin stained section;
rinsing the hematoxylin-dyed slices, and soaking the slices in hydrochloric acid alcohol with the volume fraction of 0.1% for differentiation for 3-5 s to prepare differentiated slices;
placing the differentiated slices in water to turn blue, and preparing the slices after turning blue; and
and (3) placing the slice after returning blue into absolute ethyl alcohol, then carrying out eosin dyeing treatment, and rinsing after the eosin dyeing solution is dyed, thus obtaining the hematoxylin-eosin dyed slice.
7. The method for preparing a tissue wax block according to any one of claims 1 to 3, 5 and 6, further comprising the steps of sequentially fixing, dehydrating, clearing, waxing and embedding the tissue sample to prepare a tissue wax block.
8. The method of claim 7, wherein the act of securing the tissue sample comprises:
soaking the tissue sample in a fixing solution for fixing for 24-48 h; and
and (4) washing the fixed tissue sample, and removing the fixing solution.
9. The method of claim 7, wherein the dehydrating comprises:
soaking the fixed tissue samples in the following solutions in sequence from low ethanol concentration to high ethanol concentration: 30% by volume of ethanol solution, 50% by volume of ethanol solution, 75% by volume of ethanol solution, 80% by volume of ethanol solution, 95% by volume of ethanol solution and 95% by volume of ethanol solution, wherein the soaking time of each ethanol solution is independently 1-2 h; and
soaking the tissue sample treated by the ethanol solution with the volume fraction of 95% for 2 times by using absolute ethanol.
10. The method of claim 7, wherein the act of transparentizing comprises:
and sequentially soaking the dehydrated tissue sample in a solution containing a clearing agent according to the sequence of the concentration of the clearing agent from low to high, wherein the clearing agent comprises one or more of dimethylbenzene, benzene, chloroform and n-butyl alcohol.
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