CN114279801A - Formaldehyde-free fixing solution for fixing various fresh tissues and fixing method - Google Patents
Formaldehyde-free fixing solution for fixing various fresh tissues and fixing method Download PDFInfo
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- CN114279801A CN114279801A CN202111604986.XA CN202111604986A CN114279801A CN 114279801 A CN114279801 A CN 114279801A CN 202111604986 A CN202111604986 A CN 202111604986A CN 114279801 A CN114279801 A CN 114279801A
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Abstract
The invention discloses a formaldehyde-free fixing solution for fixing various fresh tissues, which comprises a solution A and a solution B, wherein the fresh tissues are sequentially subjected to first fixing and second fixing, various substances in various tissues can be effectively fixed, the fixed tissues are not obviously expanded and contracted, the tissues keep morphological structures in a living state as much as possible, and subsequent slicing and pathological examination are facilitated.
Description
Technical Field
The invention belongs to the technical field of medical histopathology, and particularly relates to a formaldehyde-free fixing solution for fixing various fresh tissues and a fixing method.
Background
In histopathology, collected fresh tissues need to be fixed properly and effectively in time, so that autolysis and putrefaction of the tissues are prevented, intracellular components such as protein, fat, sugar, enzyme and the like are precipitated or solidified, the tissues and cells keep morphological structures in a living state as far as possible, and subsequent slicing and other work can be performed smoothly.
The fixing solution is a commonly used tissue fixing agent, the traditional tissue fixing solution is formalin (formaldehyde aqueous solution), the problems of high volatility and high toxicity of formaldehyde exist, and the environment is easily polluted and the body of an operator is damaged in the using process. Meanwhile, human tissues have various types, and different parts have different tissue structures and properties, and along with the development of immunohistochemistry and molecular biology, the fixative is required to have diversity. However, when one kind of fixative is used for each kind of human tissue, a large number of kinds of fixatives need to be prepared and marked separately, which results in a huge workload.
Ethanol is used as one kind of tissue fixing agent, has fixing effect and dehydration effect on tissue cells, when ethanol is used as the fixing agent, if high-concentration ethanol is directly adopted, the surface of a biological material is rapidly dehydrated to form a layer of structure similar to a protective film, the fixation and dehydration in the tissue are influenced, thus the concentration of the ethanol is required to have gradient (the required concentration is from high to low), the concentration gradient generally adopted in the prior art is 50% -70% -80% -95% of anhydrous ethanol, the span change among the gradients of the ethanol concentration is large, the fixation and dehydration effect is poor, if ethanol solution with small span among the gradients is used, a plurality of groups of ethanol solutions with different concentrations need to be prepared, the solution needs to be frequently changed during fixation, the preparation, fixation and dehydration process operations are complicated, and the fixing mode in the prior art is an open processing mode, when the fixing liquid needs to be replaced for many times, the sample is easy to pollute.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a formaldehyde-free fixing solution for fixing various fresh tissues, which consists of various fixing solution components, can be used for fixing the fresh tissues in sequence, can effectively fix various substances in various tissues, has unobvious expansion and contraction of the fixed tissues, ensures that the tissues keep the morphological structure in a living state as much as possible, and is beneficial to subsequent slicing and pathological examination.
The purpose of the invention is realized by the following technical scheme:
a formaldehyde-free fixing solution for fixing various fresh tissues comprises solution A and solution B;
the liquid A comprises picric acid and glacial acetic acid;
the liquid B comprises the following raw materials: glutaraldehyde and ethanol.
Preferably, the liquid B comprises liquid B1, liquid B2 and liquid B3;
the B1 liquid is prepared from the following raw materials in parts by mass: 10 parts of glutaraldehyde, 50 parts of ethanol and 40 parts of phosphate buffer;
the raw material for preparing the B2 liquid also comprises a phosphate buffer solution;
the B2 liquid is prepared from the following raw materials in parts by mass: 10 parts of glutaraldehyde, 70 parts of ethanol and 20 parts of phosphate buffer;
the B3 liquid is prepared from the following raw materials in parts by mass: 5 parts of glutaraldehyde and 95 parts of ethanol.
The glutaraldehyde is a glutaraldehyde aqueous solution, and the mass percentage content of the glutaraldehyde in the glutaraldehyde aqueous solution is 24-26%;
the concentration of the phosphate buffer solution is 0.2mol/L, and the pH value is 7.4;
the ethanol is absolute ethanol.
Preferably, the picric acid is a picric acid saturated solution, and the mass percentage of the picric acid saturated solution to the glacial acetic acid is 15: 1;
the picric acid saturated solution is based on the dosage of picric acid which can be dissolved in water to form picric acid saturated aqueous solution.
A method of preparing a formaldehyde-free fixative solution useful for fixation of a variety of fresh tissues, comprising the steps of:
step one, preparing solution A:
taking picric acid and glacial acetic acid according to mass percent, and stirring and mixing uniformly to obtain solution A;
step two, preparing liquid B:
preparing a B1 solution: taking glutaraldehyde, ethanol and a phosphate buffer solution according to the parts by weight, and uniformly stirring and mixing to obtain a B1 solution;
preparing a B2 solution: taking glutaraldehyde, ethanol and a phosphate buffer solution according to the parts by weight, and uniformly stirring and mixing to obtain a B2 solution;
preparing a B3 solution: taking glutaraldehyde and ethanol according to the parts by weight, stirring and mixing uniformly to prepare B3 liquid.
Preferably, the method for fixing fresh tissue by using the fixing solution comprises the following steps:
s1, placing fresh tissues into the solution A, and carrying out first fixation for 3-5 hours;
when the first fixation is carried out, the volume ratio of the solution A to the tissue to be fixed is 10-30: 1, preferably 20 to 30: 1;
s2, after the first fixation is finished, washing tissues by using PBS buffer solution;
s3, placing the cleaned tissue in a fixing chamber, adding the solution B, and performing second fixing;
and when the second fixation is carried out, the volume ratio of the solution B to the tissue to be fixed is 10-30: 1, preferably 20 to 30: 1.
preferably, the step of adding liquid B includes replacement of a portion of the used liquid.
Preferably, the addition sequence of the solutions in the solution B in the S3 is as follows: adding the B1 solution, then adding the B2 solution and finally adding the B3 solution.
Preferably, in the process of performing the second fixing in S3, the following three liquid changing modes are sequentially performed:
a first liquid changing mode, when B2 liquid needs to be added into the fixed chamber, if the liquid in the current fixed chamber is used B1 liquid, part of the liquid in the current fixed chamber is discharged, and B2 liquid is added to carry out first liquid changing;
a second liquid changing mode, when B2 liquid needs to be added into the fixed chamber, if the liquid in the current fixed chamber comprises the used B2 liquid, part of the liquid in the current fixed chamber is discharged, and B2 liquid is added to carry out second liquid changing;
in the third liquid changing mode, when B3 liquid needs to be added into the fixed chamber, part of the liquid in the fixed chamber is discharged and B3 liquid is added, and the third liquid changing is carried out.
The mass percentage of the ethanol in the liquid in the fixed chamber is gradually increased along with the addition times of the B2 liquid and the B3 liquid.
Preferably, there is linkage room above the fixed room, there is a movable piston between linkage room and fixed room, when the movable piston is closed, can move up and down between linkage room and fixed room in a sealed way, move up and down the movable piston in order to change the volume of fixed room, linkage room;
the replacement of the partially used liquid comprises the following steps:
when it is necessary to replace a part of the liquid in the holding chamber,
s101, filling new liquid into the linkage chamber, descending the movable piston in a force accumulation mode, driving the linkage assembly to move to the opening of the liquid discharge hole, and discharging part of the used liquid in the fixed chamber from the liquid discharge hole in an opening state;
s102, after the piston descends to a set position along with the movement, the linkage assembly is driven to move until the liquid discharge hole is closed again;
s103, the movable piston is opened, so that the empty chamber of the movable piston is exposed, the fixed chamber, the linkage chamber and the empty chamber are communicated, new liquid is poured into the empty chamber, the stored force starts to be released, the liquid discharge hole is kept closed while the movable piston moves in a resetting mode until the movable piston is reset, the movable piston is closed, the linkage chamber and the fixed chamber are sealed and separated again, and replacement of part of used liquid is completed.
Preferably, the linkage assembly comprises linkage strip-shaped teeth and transmission ratchets; the upper end of the linkage bar-shaped tooth is rotationally matched with the movable piston; the linkage strip-shaped teeth are in one-way linkage with the transmission ratchets;
the linkage bar-shaped tooth up-and-down reciprocating motion comprises the following steps:
step one, when the linkage bar-shaped teeth move downwards, the transmission ratchets are contacted to drive the transmission ratchets to rotate in a force storage mode so as to open the liquid discharge hole;
step two, when the linkage bar-shaped teeth continue to move downwards to be separated from the transmission ratchet, the transmission ratchet releases the stored force to reset and rotate, and the liquid discharge hole is closed;
and step three, in the process of upward resetting of the linkage strip-shaped teeth, the reverse rotation of the transmission ratchets is limited due to unidirectional blocking, and the linkage strip-shaped teeth overcome the blocking of the transmission ratchets in a deflection mode and reset upward.
The invention has the following effects:
picric acid: is an acidic fixing agent, can generate precipitation effect on all proteins, and has obvious contraction effect;
glacial acetic acid: can precipitate nucleoprotein and can better maintain the chromosome structure;
glutaraldehyde: is an aldehyde with double functions, has better fixing function on glycogen, glycoprotein, microtubule endoplasmic reticulum, cell matrix and the like, and can well preserve tissues;
ethanol: is a lipid solvent capable of dissolving fat and lipoid, and can be soaked for a long time to harden tissue, deform cell nucleus, shrink cytoplasm, slowly denature protein, and precipitate glycogen.
Compared with the prior art, the invention has the following beneficial effects:
the fixing liquid of the invention does not contain formaldehyde, thus avoiding the pollution of formaldehyde to the air environment and the health threat of operators and related personnel in the using process, and the stimulation of glutaraldehyde is much less than that of formaldehyde under the same condition, thus having higher environmental protection and safety. Firstly, preparing solution A by picric acid and glacial acetic acid to carry out first fixing treatment on fresh tissues; the glacial acetic acid has a strong penetrating effect, so that the tissue can be fixed in a short time, and then the glutaraldehyde and ethanol are used for further fixing the tissue, so that the defect of weak penetrating force of the glutaraldehyde is effectively overcome, the tissue is quickly fixed, the fixed tissue is not obviously expanded and contracted, the tissue keeps a morphological structure in a living state as much as possible, and subsequent slicing and pathological examination are facilitated.
The picric acid and the glacial acetic acid are matched for use, so that the tissue structure can be well preserved, the picric acid can generate precipitation effect on all proteins, but has contraction effect on the tissues, the glacial acetic acid can resist excessive tissue contraction caused by other fixing agents such as the picric acid and the like while rapidly precipitating nucleoprotein and well keeping the chromosome structure, and the picric acid can also soften the tissues, prevent the tissues from being excessively hardened and is not beneficial to subsequent slicing;
glutaraldehyde and ethanol are used for further fixing tissues, the glutaraldehyde has a good fixing effect on glycogen, glycoprotein, microtubule endoplasmic reticulum, cell matrix and the like, the ethanol can precipitate albumin, globulin and nucleoprotein, the ethanol has a fixing effect and a dehydrating effect on tissue cells, the ethanol with a concentration from low to high is used in cooperation with the glutaraldehyde, continuous gradient dehydration of the tissues is realized while the tissues are fixed, the phenomenon that the surface of a biological material is rapidly dehydrated to form a structure similar to a protective film by directly dehydrating with high-concentration ethanol, the dehydration of an internal material is influenced is avoided, the tissues after second fixing treatment can be subjected to subsequent treatments such as transparency, embedding, slicing and the like only by finally performing dehydration treatment with absolute ethanol, the tissue fixing and tissue dehydration are performed synchronously, and the efficiency of pathological examination is effectively improved;
in addition, the picric acid in the solution A used for the first fixation can yellow the tissues, and the problem can be effectively solved after the treatment of the second fixation ethanol;
according to the invention, gradient difference exists from low to high in the mass percentage of ethanol in the solution B from the solution B1 (the mass percentage of ethanol is 50%), the solution B2 (the mass percentage of ethanol is 70%) and the solution B3 (the mass percentage of ethanol is 95%), and the gradient span of the mass percentage of ethanol in the liquid in the fixing chamber is reduced through treatment of the liquid after part replacement, so that gradient slow fixation and dehydration of ethanol on tissues can be effectively realized, and the dehydration effect is good.
Drawings
FIG. 1 is a flow chart of a process for preparing interstitial fluid according to the present invention;
FIG. 2 is a flow chart of the present invention for fixation of fresh tissue using interstitial fluid;
FIG. 3 is a schematic view of the overall three-dimensional internal structure of the tissue fastening device of the present invention;
FIG. 4 is a schematic view of the overall planar internal structure of the tissue fastening device of the present invention;
FIG. 5 is a second schematic view of the overall three-dimensional internal structure of the tissue fastening device of the present invention;
FIG. 6 is a schematic illustration of an exploded view of the moving piston portion of the tissue fastening device of the present invention;
FIG. 7 is a schematic view of a portion of the linkage assembly of the tissue fastening device of the present invention;
FIG. 8 is a second schematic view of a portion of the linkage assembly of the tissue fastening device of the present invention;
FIG. 9 is an enlarged view of a portion of FIG. 8A;
FIG. 10 is a schematic view of the drive rack and pinion of the tissue fastening device of the present invention.
In the figure: 1. a liquid tank; 11. a fixed chamber; 111. a guide bar; 12. a linkage chamber; 121. a water inlet pipe; 122. a control valve; 123. a baffle plate; 13. a pressure relief wall; 14. a pH value detector; 2. moving the piston; 21. a cavity; 22. an opening; 23. moving the frame; 231. an electric push rod; 24. an empty chamber; 25. a support bar; 3. a reset assembly; 31. a limiting cylinder; 32. a compression spring; 4. a drain hole; 41. fixing the rod; 5. a linkage assembly; 51. linkage bar-shaped teeth; 511. a wedge-shaped projection; 52. a linkage gear set; 521. a drive ratchet; 522. a drive gear; 523. a sector sealing block; 524. a fan-shaped through hole; 525. a torsion spring; 526. a fixing ring; 527. a transmission gear.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without any creative work based on the embodiments of the present invention belong to the protection scope of the present invention.
Example 1
Preparation of fixative solutions for various fresh tissue fixations:
weighing the following raw materials:
weighing the raw materials for preparing the solution A according to the mass percentage: 15: 1, picric acid saturated solution and glacial acetic acid;
raw materials for preparing the solution B:
weighing the following raw materials for preparing the B1 liquid in parts by weight: 10 parts of glutaraldehyde aqueous solution with the mass percentage of 25% (namely the mass percentage of the glutaraldehyde in the glutaraldehyde aqueous solution is 25%), 40 parts of absolute ethyl alcohol and 50 parts of phosphate buffer;
weighing the following raw materials for preparing the B2 liquid in parts by weight: 10 parts of glutaraldehyde aqueous solution with the mass percentage of 25% (namely the mass percentage of the glutaraldehyde in the glutaraldehyde aqueous solution is 25%), 70 parts of absolute ethyl alcohol and 20 parts of phosphate buffer;
weighing the following raw materials for preparing the B3 liquid in parts by weight: 5 parts of glutaraldehyde aqueous solution with the mass percentage of 25% (namely the mass percentage of the glutaraldehyde in the glutaraldehyde aqueous solution is 25%), and 95 parts of absolute ethyl alcohol;
the method comprises the following steps:
s1, preparing a solution A: stirring and mixing the picric acid saturated solution and glacial acetic acid uniformly to obtain solution A;
s2, preparing a liquid B:
preparing a B1 solution: uniformly stirring and mixing glutaraldehyde aqueous solution, absolute ethyl alcohol and phosphate buffer solution to obtain B1 solution;
preparing a B2 solution: uniformly stirring and mixing glutaraldehyde aqueous solution, absolute ethyl alcohol and phosphate buffer solution to obtain B2 solution;
preparing a B3 solution: and uniformly stirring and mixing the glutaraldehyde aqueous solution and the absolute ethyl alcohol to obtain a B3 solution.
Example 2
The tissue fluid prepared in example 1 was used for fixation of fresh tissue of the breast, comprising the following steps:
s1, placing fresh tissue of a mammary gland into liquid A, wherein the volume ratio of the liquid A to the fresh tissue of the mammary gland is 20: 1, carrying out first fixation for 3 hours;
s2, after the first fixation is finished, cleaning the mammary tissue by using PBS buffer solution;
s3, placing the cleaned breast tissue in a fixing chamber, adding liquid B, and performing second fixing, wherein the volume ratio of the liquid B to the fresh tissue of the breast is 20: 1;
the method specifically comprises the following steps: firstly, filling the B1 liquid into a fixing chamber in which the cleaned mammary tissue is placed for fixing;
after 3 hours of the fixing treatment, the liquid after partial use was replaced by injecting a new B2 liquid, and every 1 hour of the fixing treatment, the liquid after partial use was replaced by injecting a new B2 liquid for a total of 5 times;
after the partially used liquid was replaced by injecting a new B2 liquid 5 times, the fixation treatment was carried out for 1 hour, and the partially used liquid was replaced by injecting a new B3 liquid, and the partially used liquid was replaced by injecting a new B3 liquid every 0.5 hour of the fixation treatment for 5 times in total.
The total fixation time of the first fixation and the second fixation was 13.5 hours.
Example 3
The tissue fluid prepared in example 1 is used for fixing fresh tissues of thyroid gland, and comprises the following steps:
s1, placing a fresh group of thyroid in liquid A, wherein the volume ratio of the liquid A to fresh tissue of the thyroid is 20: 1, carrying out first fixation for 3 hours;
s2, after the first fixation is finished, cleaning thyroid tissue by using PBS buffer solution;
s3, placing the cleaned thyroid tissue in a fixing chamber, adding liquid B, and performing second fixing, wherein the volume ratio of the liquid B to the fresh thyroid tissue is 20: 1;
the method specifically comprises the following steps: filling a B1 liquid into a fixing chamber in which the cleaned thyroid tissue is placed for fixing;
after 3 hours of the fixing treatment, the liquid after partial use was replaced by injecting a new B2 liquid, and every 1 hour of the fixing treatment, the liquid after partial use was replaced by injecting a new B2 liquid for a total of 5 times;
after the partially used liquid was replaced with the 5 th new B2 liquid, the fixation treatment was carried out for 1 hour, the partially used liquid was replaced with the new B3 liquid, and the partially used liquid was replaced with the new B3 liquid every 1 hour of the fixation treatment for 5 times in total.
The total fixation time of the first fixation and the second fixation was 16 hours.
Example 4
The tissue fluid prepared in example 1 is used for fixing fresh tissues of an ovary, and comprises the following steps:
s1, placing fresh ovary tissues in solution A, wherein the volume ratio of the solution A to the fresh ovary tissues is 20: 1, carrying out first fixation for 3 hours;
s2, after the first fixation is finished, washing ovarian tissues by using PBS buffer solution;
s3, placing the washed ovary tissue into a fixing chamber, adding liquid B, and performing second fixing, wherein the volume ratio of the liquid B to the fresh ovary tissue is 20: 1;
the method specifically comprises the following steps: firstly, filling the B1 liquid into a fixing chamber in which the washed ovarian tissues are placed for fixing;
after 3 hours of fixation treatment, injecting new B2 liquid to replace the partially used liquid, and injecting new B2 liquid to replace the partially used liquid every 0.5 hours of fixation treatment for 5 times;
after the partially used liquid was replaced with the 5 th new B2 liquid, the fixation treatment was carried out for 0.5 hour, and the partially used liquid was replaced with the new B3 liquid, and the partially used liquid was replaced with the new B3 liquid every 1 hour of the fixation treatment for 5 times.
The total fixation time of the first fixation and the second fixation was 13.5 hours.
In examples 2 to 4, the volume of the liquid to be replaced was 20% of the total volume of the liquid in the holding chamber every time the replacement of the liquid after the partial use was performed.
Comparative example 1
Placing the fresh tissue of the mammary gland into 95% ethanol water solution by mass percentage (namely, the mass percentage of ethanol in the ethanol water solution is 95%) for fixation, wherein the volume ratio of the 95% ethanol water solution to the fresh tissue of the mammary gland is 20: the fixation time of the mammary tissue was the same as the total fixation time of the first and second fixations in example 2, i.e. 13.5 hours.
Comparative example 2
Placing fresh thyroid tissue into 95% ethanol water solution (namely, ethanol accounts for 95% of the ethanol water solution by mass) for fixation, wherein the volume ratio of the 95% ethanol water solution to the fresh mammary tissue is 20: the time for fixing the thyroid tissue was the same as the total fixing time of the first and second fixations in example 3, i.e., 16 hours.
Comparative example 3
Placing fresh ovary tissues into 95% ethanol water solution (namely, the mass percentage of ethanol in the ethanol water solution is 95%) for fixation, wherein the volume ratio of the 95% ethanol water solution to the fresh breast tissues is 20: the fixation time of ovarian tissue was 13.5 hours as the total fixation time of the first and second fixations in example 4.
Comparative example 4
Placing the fresh tissue of the mammary gland into a 2.5 mass percent pentanediol solution (prepared by 10 parts of 25 mass percent pentanediol water solution (the mass percent of glutaraldehyde in the glutaraldehyde water solution is 25%) and 90 parts of phosphate buffer) for fixation, wherein the volume ratio of the 2.5 mass percent pentanediol solution to the fresh tissue of the mammary gland is 20: the fixed time is the same as the total fixed time of the first and second fixing in example 2, i.e. 13.5 hours.
Example 5
This example discloses a tissue fixation device that can be used to replace a portion of the fluid used during the first fixation of example 4.
When the liquid B is used for fixing tissues, the mass percentage of ethanol contained in the liquid in the fixing chamber is gradually increased by replacing part of the used liquid in the fixing chamber, the gradient fixation and dehydration of the ethanol on the tissues are favorably realized, and the fixation and dehydration effects are good.
Referring to fig. 3, a fluid changing apparatus for fixing fresh tissue includes a fluid tank 1, the fluid tank 1 includes a fixing chamber 11 and a linkage chamber 12, and a used fluid in the fixing chamber 11 is discharged and refreshed by injecting a new fluid into the linkage chamber 12; a movable piston 2 is arranged between the fixed chamber 11 and the linkage chamber 12, a cavity 21 is arranged in the movable piston 2, an opening 22 is formed in the lower surface of the movable piston 2, a movable frame 23 is arranged at the position, located at the opening 22, of the cavity 21, and the opening 22 can be sealed by the movable frame 23; when a new liquid is injected into the linkage chamber 12, the movable piston 2 moves toward the fixed chamber 11, and the same amount of used liquid is discharged from the fixed chamber 11; after a certain amount of new liquid is injected into the linkage chamber 12, the liquid discharge of the fixed chamber 11 is stopped, the movable frame 23 in the cavity 21 moves, the opening 22 is opened, a hollow chamber 24 is formed, and the hollow chamber 24 penetrates through the movable piston 2; the bottom of the movable piston 2 is provided with a reset component 3, and the reset component 3 is used for resetting the movable piston 2; one side of the bottom of the fixed chamber 11 is provided with a liquid discharge hole 4, the liquid discharge hole 4 comprises a linkage component 5, the linkage component 5 is matched with the movable piston 2 to be used for opening and closing the liquid discharge hole 4, and the side wall of the bottom of the fixed chamber 11 is provided with a pressure relief wall 13.
Referring to fig. 4, further, a water inlet pipe 121 is disposed on one side above the linkage chamber 12, a control valve 122 is disposed between the water inlet pipe 121 and the linkage chamber 12, a baffle 123 is disposed on the top of an inner cavity of the linkage chamber 12, so that a liquid injection space is left on the top of the linkage chamber 12, and a new liquid is injected into the linkage chamber 12 by connecting the water inlet pipe 121 to an external fixed liquid injection device, wherein the control valve 122 is disposed to cooperate with an injection device, when a new liquid needs to be injected into the linkage chamber 12, the control valve 122 is opened, the external new liquid is injected into the linkage chamber 12 through the water inlet pipe 121 and the control valve 122, after the injection is completed, the injection device stops working, the control valve 122 is simultaneously closed, and the linkage chamber 12 is cut off from the outside, so that the linkage chamber 12 is in a closed state;
a communicating valve is arranged on one side above the linkage chamber 12, so that new liquid can be conveniently injected into the linkage chamber 12 for the first time; a placing opening for conveniently placing fresh tissues is arranged at the front side of the fixing chamber 11, and a sealing part is arranged at the placing opening, which is the prior art and is not described again;
in the actual operation process, when liquid is injected into the linkage chamber 12 for the first time, the baffle 123 is arranged at the top of the linkage chamber 12, so that a certain space is reserved above the movable piston 2, and then the space at the baffle 123 is filled with new liquid after the liquid tank 1 is filled with the new liquid.
Referring to fig. 4-6, further, the opening 22 is located in the middle of the upper portion of the cavity 21, the moving frames 23 are respectively located on two sides of the opening 22, an electric push rod 231 is disposed on the outer side of each moving frame 23, the electric push rod 231 is fixed inside the cavity 21, an output shaft of the electric push rod 231 is connected to the moving frame 23, support rods 25 are disposed on two sides of the electric push rod 231, the support rods 25 are used for supporting the cavity 21, the moving frames 23 are driven by the electric push rod 231 to move, so that the moving frames 23 on two sides are combined and separated, and in the moving process, the support rods 25 support the cavity 21, so that the inside of the cavity 21 is more stable.
Further, the electric push rod 231 drives the moving frame 23 to move, and when the two moving frames 23 are combined, the opening 22 is closed; when the two moving frames 23 are separated, the space of the formed empty chamber 24 is enlarged, so that new liquid in the linkage chamber 12 flows into the empty chamber 24, the pressure of the liquid above the fixed chamber 11 on the moving piston 2 is reduced, the empty chamber 24 is formed at the opening 22 through the separation of the two moving frames 23, the space of the moving piston 2 is enlarged, the empty chamber 24 is supplemented to the liquid in the linkage chamber 12 due to the pressure, the pressure given to the moving piston 2 by the liquid in the linkage chamber 12 is reduced, the balance state of the moving piston 2 is broken, at the moment, under the action of the resetting component 3, the moving piston 2 continuously moves upwards, the space in the linkage chamber 12 is compressed, the liquid in the linkage chamber 12 flows into the fixed chamber 11 through the empty chamber 24, and the supplement of the liquid in the fixed chamber 11 is completed.
Further, the reset assembly 3 includes a limiting cylinder 31, a compression spring 32 is disposed inside the limiting cylinder 31, when the movable piston 2 is located at the top of the linkage chamber 12, the compression spring 32 is in a semi-compressed state, two ends of the compression spring 32 are respectively connected with the inner wall of the limiting cylinder 31 and the bottom of the inner cavity of the fixed chamber 11, a guide rod 111 is disposed at the bottom of the fixed chamber 11, the guide rod 111 is located inside the compression spring 32, when the movable piston 2 is located at the top of the linkage chamber 12 through the compression spring 32 in the semi-compressed state, the movable piston 2 is always subjected to upward elastic force of the compression spring 32, after the movable piston 2 moves downward for a certain distance, the compression spring 32 is further compressed, so that elastic potential energy possessed by the compression spring 32 is further increased, when the linkage chamber 12 is communicated with the fixed chamber 11 and a pressure difference is formed between the linkage chamber 12 and the fixed chamber 11, the compression spring 32 pushes the movable piston 2 to move upward, the new liquid in the linkage chamber 12 is injected into the fixed chamber 11 through the empty chamber 24, wherein when the movable piston 2 moves on the line, the space of the fixed chamber 11 expands, and the space of the linkage chamber 12 compresses, so that the new liquid in the linkage chamber 12 is injected into the fixed chamber 11.
Referring to fig. 7-8, further, the linkage assembly 5 includes a linkage bar-shaped tooth 51 and a linkage gear set 52, the linkage bar-shaped tooth 51 is used for driving the linkage gear set 52, the linkage bar-shaped tooth 51 is rotatably connected to one side of the bottom of the movable piston 2, a wedge-shaped protrusion 511 is disposed at a position of the bottom of the fixed chamber 11, which is opposite to the vertical state of the linkage bar-shaped tooth 51, an inclined surface of the wedge-shaped protrusion 511 is located at one side of teeth of the linkage bar-shaped tooth 51, the linkage bar-shaped tooth 51 moves downward under the driving of the movable piston 2 through the linkage bar-shaped tooth 51, and when the bottom of the linkage bar-shaped tooth 51 moves to the position of the wedge-shaped protrusion 511, the linkage bar-shaped tooth 51 is deflected to the outside, and the teeth of the linkage bar-shaped tooth 51 are further far away from the linkage gear set 52.
Referring to fig. 9-10, further, the linkage gear set 52 includes a transmission ratchet 521, the transmission ratchet 521 is located below one side of the linkage bar-shaped tooth 51, the transmission ratchet 521 is meshed with the linkage bar-shaped tooth 51, a transmission gear 527 is coaxially and fixedly disposed on one side of the transmission ratchet 521, a drive gear 522 is meshed with the other side below the transmission gear 527, a sector seal block 523 is disposed on the back of the drive gear 522, the transmission ratchet 521 and the drive gear 522 are both rotatably connected to the inner wall of the fixed chamber 11, the transmission ratchet 521 is meshed with the linkage bar-shaped tooth 51 through the transmission ratchet 521, so that the transmission ratchet 521 is driven to rotate when the linkage bar-shaped tooth 51 moves downward, the transmission gear 527 on the other side of the transmission ratchet 521 rotates in the same direction, the drive gear 522 meshed with the transmission gear 527 is driven to rotate, and when the drive gear 522 rotates, the sector seal block 523 is driven to release the drain hole 4;
when the linkage bar-shaped teeth 51 move downwards, the ratchet teeth on the linkage bar-shaped teeth 51 and the ratchet teeth on the transmission ratchet 521 are limited mutually, so that the linkage bar-shaped teeth 51 are meshed with the transmission ratchet 521 all the time; when the linkage bar-shaped tooth 51 moves upwards, one inclined surface of the tooth on the linkage bar-shaped tooth 51 and one inclined surface of the ratchet tooth on the transmission ratchet 521 repel each other, so that the linkage bar-shaped tooth 51 deflects outwards when moving upwards, and the linkage bar-shaped tooth 51 cannot transmit with the transmission ratchet 521.
Further, when the interlocking bar-shaped teeth 51 move downward, the frictional force between the ratchet teeth on the interlocking bar-shaped teeth 51 and the ratchet teeth on the transmission ratchet 521 is greater than the lateral thrust force applied to the interlocking bar-shaped teeth 51 by the transmission ratchet 521, and the interlocking bar-shaped teeth 51 do not deflect outward.
Further, a second torsion spring is arranged between the linkage bar-shaped tooth 51 and the movable piston 2, and when the linkage bar-shaped tooth 51 moves downwards, the linkage bar-shaped tooth 51 is prevented from deflecting by the elastic limit of the second torsion spring; in the process of resetting the linked bar-shaped teeth 51 upwards, the reset of the linked bar-shaped teeth 51 in the deflection state can be realized by the reset of the arranged second torsion spring.
Referring to fig. 9, further, a sector sealing block 523 is used for blocking the liquid discharge hole 4, a sector through hole 524 is disposed at one side of the sector sealing block 523, the sector through hole 524 is used for communicating the liquid discharge hole 4 with the fixed chamber 11, a fixing rod 41 is disposed at one side of the liquid discharge hole 4, the fixing rod 41 is located inside the sector through hole 524, the fixing rod 41 cooperates with the sector through hole 524 to limit the rotation range of the driving gear 522, and the sector sealing block 523 cooperates with the sector through hole 524 at the other side to achieve blocking and communicating between the liquid discharge hole 4 and the fixed chamber 11 under the driving of the driving gear 522.
Furthermore, the back of the driving gear 522 is further provided with a torsion spring 525, the outer sides of the fan-shaped sealing block 523 and the fan-shaped through hole 524 are provided with a fixing ring 526, the fixing ring 526 is fixed on the side wall of the fixing chamber 11, two ends of the torsion spring 525 are respectively connected with the driving gear 522 and the fixing ring 526, through the arranged torsion spring 525, when the driving gear 522 is driven by the transmission gear 527 to rotate, the torsion spring 525 deforms and condenses elastic potential energy, and when the driving gear 522 loses the power of the transmission gear 527, the torsion spring 525 releases the elastic potential energy, so that the driving gear 522 reversely rotates and recovers;
under the action of the fixing rod 41 arranged in the fan-shaped through hole 524, the fan-shaped sealing block 523 and the fan-shaped through hole 524 can rotate to the maximum extent, only the upper and lower positions of the fan-shaped sealing block 523 and the fan-shaped through hole 524 can be interchanged, and the driving gear 522 is reversely rotated and restored under the action of the torsion spring 525, namely when the drain hole 4 is blocked by the fan-shaped sealing block 523, the driving gear 522 cannot continue to rotate reversely, at the moment, when the linkage bar-shaped tooth 51 moves upwards, the reverse rotation of the driving gear 522 is blocked due to the existence of the fixing rod 41, so that the driving gear 522 cannot continue to rotate reversely, transmission ratchets cannot be generated between the linkage bar-shaped tooth 51 and the transmission ratchets 521, the linkage bar-shaped tooth 51 is rotated outwards when moving upwards under the action force of the transmission 521, and then the linkage bar-shaped tooth 51 is reset independently.
In use, fresh tissue to be fixedly stored is first placed inside the fixing chamber 11 through the placing opening at the front side of the fixing chamber 11.
Injecting liquid for the first time: the two moving frames 23 are separated through the electric push rod 231, so that the opening 22 above the moving piston 2 is in an open state, then whether the moving piston 2 is positioned at the uppermost part of the inner cavity of the liquid tank 1 or not is checked, and after the moving piston 2 is positioned at the uppermost part of the inner cavity of the liquid tank 1, the sector sealing block 523 blocks the liquid discharge hole 4, so that the liquid discharge hole 4 is in a closed state; further opening the control valve 122 on one side above the linkage chamber 12, controlling the injection of new liquid into the linkage chamber 12, opening the communication valve on the other side above the linkage chamber 12, so that new liquid can flow into the fixed chamber 11 through the empty chamber 24 when the liquid is injected for the first time until the liquid is filled in the liquid tank 1, wherein the formed empty chamber 24 is included, the air in the liquid tank 1 is discharged through the communication valve, when a small amount of liquid overflows from the communication valve, closing the control valve 122 at the position of the water inlet pipe 121, at this time, driving the two moving frames 23 to merge through the electric push rod 231 again, so that the opening 22 above the moving piston 2 is closed, namely the empty chamber 24 is closed, the liquid in the empty chamber 24 is discharged and collected, and then closing the communication valve, and completing the injection for the first time;
in the practical process, when liquid is injected into the linkage chamber 12 for the first time, the baffle 123 is arranged at the top of the linkage chamber 12, so that a certain space is reserved above the movable piston 2, and the space at the baffle 123 is filled with new liquid after the liquid tank 1 is filled with the new liquid;
liquid changing: firstly, the control valve 122 at the water inlet pipe 121 is opened, new liquid with a higher pH value is injected into the linkage chamber 12, a certain space is reserved above the movable piston 2 due to the baffle 123 arranged at the top of the linkage chamber 12, when new liquid is injected into the linkage chamber 12, the liquid in the space at the baffle 123 is further in a closed state at the moment, the opening 22 above the movable piston 2 is in a closed state, when the new liquid is injected into the linkage chamber 12, the movable piston 2 receives pressure and conducts the liquid in the fixed chamber 11, after the liquid in the fixed chamber 11 receives the pressure, the pressure relief wall 13 at one side of the fixed chamber 11 is expanded outwards, the movable piston 2 can move downwards, when the movable piston 2 moves downwards, the linkage bar-shaped teeth 51 are driven to move downwards, when the linkage bar-shaped teeth 51 move downwards, the ratchet teeth on the linkage bar-shaped teeth 51 and the ratchet teeth on the transmission ratchet teeth 521 are limited mutually, the linkage bar-shaped teeth 51 are always meshed with the transmission ratchets 521, the transmission ratchets 521 are meshed with the linkage bar-shaped teeth 51 through the transmission ratchets 521, the linkage bar-shaped teeth 51 are driven to rotate when moving downwards, then the transmission gears 527 on the other sides of the transmission ratchets 521 rotate in the same direction, and the driving gears 522 meshed with the transmission gears 527 are driven to rotate, in the process, the torsion springs 525 arranged on the backs of the driving gears 522 are deformed to condense elastic potential energy, and when the driving gears 522 rotate, the sector sealing blocks 523 are driven to release the liquid discharge holes 4; the used liquid in the fixed chamber 11 is discharged from the liquid discharge hole 4, when the movable piston 2 moves downwards until the limiting cylinder 31 abuts against the bottom surface of the inner cavity of the fixed chamber 11, the compression spring 32 is compressed into the limiting cylinder 31, the bottom of the linkage strip-shaped tooth 51 moves to the position of the wedge-shaped bump 511 at the moment, due to the action of the wedge-shaped bump 511, the linkage strip-shaped tooth 51 deflects outwards, the tooth part of the linkage strip-shaped tooth 51 is further far away from the linkage gear set 52, the driving gear 522 in the linkage gear set 52 is not limited, when the driving gear 522 loses the power of the transmission gear 527, the torsion spring 525 releases elastic potential energy, the driving gear 522 reverses and recovers, at the moment, the driving gear 522 reverses, the upper and lower positions of the fan-shaped sealing block 523 and the fan-shaped through hole 524 are interchanged, and the fan-shaped sealing block 523 blocks the liquid discharge hole 4, and closes the control valve 122;
then, the electric push rod 231 drives the moving frame 23 to move, so that the moving frames 23 on the two sides are combined and separated, a hollow chamber 24 is formed at the opening 22, the space of the moving piston 2 is enlarged, the hollow chamber 24 is supplemented by liquid in the linkage chamber 12 due to pressure, the pressure applied to the moving piston 2 by the liquid in the linkage chamber 12 is reduced, the balance state of the moving piston 2 is broken, at the moment, under the action of the reset component 3, the moving piston 2 moves upwards all the time, the space in the linkage chamber 12 is compressed, the liquid in the linkage chamber 12 flows into the fixed chamber 11 through the hollow chamber 24, the supplementation of the liquid in the fixed chamber 11 is completed, after the moving piston 2 is pushed to the top of the linkage chamber 12, the electric push rod 231 drives the moving frame 23 to move, the hollow chamber 24 is closed, and the liquid in the hollow chamber 24 is extruded to the top of the linkage chamber 12;
when the movable piston 2 is positioned at the top of the linkage chamber 12, the movable piston 2 is always subjected to upward elastic force of the compression spring 32 through the compression spring 32 in a semi-compressed state, after the movable piston 2 moves downwards for a certain distance, the compression spring 32 is further compressed, so that the elastic potential energy of the compression spring 32 is further increased, when the linkage chamber 12 is communicated with the fixed chamber 11 and the gravity above the movable piston 2 is reduced, the compression spring 32 pushes the movable piston 2 to move upwards, new liquid in the linkage chamber 12 is injected into the fixed chamber 11 through the empty chamber 24, when the movable piston 2 moves on the line, the linkage chamber 12 is compressed in space, the fixed chamber 11 is expanded in space, and the new liquid in the linkage chamber 12 is injected into the fixed chamber 11;
when moving piston 2 upward movement, upward movement is also followed to linkage bar tooth 51, because tooth on the linkage bar tooth 51 is the right angle tooth, and the right angle is located the upside, the ratchet tooth on the transmission ratchet 521 is the right angle tooth, and the right angle is located the downside, when linkage bar tooth 51 upward movement, the oblique one side of the ratchet tooth on the one side of the transmission ratchet 521 on the linkage bar tooth 51 and the transmission ratchet 521 repel each other, outwards deflect when making linkage bar tooth 51 upward movement, linkage bar tooth 51 can't transmit with transmission ratchet 521, when avoiding linkage bar tooth 51 upward movement, lead to influencing the leakproofness of outage 4.
Further, the difference from the above is that a driving tooth is arranged on the linkage bar-shaped tooth 51, a linkage tooth is arranged on one side of the transmission ratchet 521 separately, a sector-shaped tooth is arranged on the other side of the transmission ratchet 521, the driving tooth on the linkage bar-shaped tooth 51 is matched with the linkage tooth on the transmission ratchet 521, the driving gear 522 is arranged on the other side of the transmission ratchet 521, the sector-shaped tooth on the transmission ratchet 521 is meshed with the driving gear 522, and when the linkage bar-shaped tooth 51 drives the transmission ratchet 521 to rotate once, the driving gear 522 drives the sector-shaped sealing block 523 to complete the work of opening the liquid discharge hole 4 once.
When the linkage bar-shaped teeth 51 move downwards until the driving teeth and the linkage teeth are completely staggered, the transmission ratchet 521 resets to close the liquid discharge hole 4 under the resetting action of the torsion spring 525. When the linkage bar-shaped teeth 51 are reset upwards, the existence of the fixing rod 41 blocks the driving gear 522 from reversing, so that the driving gear 522 cannot continuously reverse, transmission cannot be generated between the linkage bar-shaped teeth 51 and the transmission ratchets 521, the linkage bar-shaped teeth 51 rotate outwards while moving upwards under the action of the transmission ratchets 521, and further the independent reset of the linkage bar-shaped teeth 51 is realized.
Further, when the linkage bar-shaped teeth 51 move downwards and the linkage bar-shaped teeth 51 are matched with the wedge-shaped bumps 511, the linkage bar-shaped teeth 51 rotate to be separated from the transmission ratchet 521 under the action of the wedge-shaped bumps 511, and the driving gear 522 completes rotation resetting under the action of the torsion spring 525.
Preferably, a pH detector 14 is also provided on the side of the stationary chamber 11 in the apparatus, and a controller is used in conjunction with the apparatus to determine the pH in the liquid in the stationary chamber 11.
Test examples
The breast tissue after the fixation treatment with the fixing solution prepared in example 1 in example 2, the thyroid tissue after the fixation treatment with the fixing solution prepared in example 1 in example 3, the ovarian tissue after the fixation treatment with the fixing solution prepared in example 1 in example 4, the breast tissue after the fixation treatment with a 95% ethanol aqueous solution (i.e., 95% by mass of ethanol in an ethanol aqueous solution) in comparative example 1, the thyroid tissue after the fixation treatment with a 95% ethanol aqueous solution (i.e., 95% by mass of ethanol in an ethanol aqueous solution) in comparative example 2, the ovarian tissue after the fixation treatment with a 95% ethanol aqueous solution (i.e., 95% by mass of ethanol in an ethanol aqueous solution) in comparative example 3, the breast tissue after the fixation treatment with a 2.5% glutaraldehyde solution in comparative example 4, were dehydrated, and, The morphology of the tissue was observed by treatments such as transparency, embedding, and sectioning, and the morphology of each treated tissue was recorded as shown in table 1.
TABLE 1
The table 1 shows that the formaldehyde-free fixing solution for fixing various fresh tissues can be used for fixing various fresh tissues, the tissues fixed by the fixing solution have no obvious shrinkage, the hardness is moderate, the sections are complete, no cracks exist, the structure is clear, the layers are distinct, the pathological observation of the tissues is facilitated, the uniform dehydration of the tissues is realized while the tissues are fixed, the subsequent dehydration time is shortened, and the efficiency of pathological examination is improved.
In comparative examples 1 to 3, 95% ethanol aqueous solution (i.e., ethanol in 95% by mass in ethanol aqueous solution) was used for fixation, and since ethanol dehydration was strong, the tissue after fixation contracted and hardened, affecting the quality of the section, and not beneficial to the pathological examination of the tissue, and since high concentration ethanol was used for fixation, the tissue surface was rapidly dehydrated to form a structure similar to a protective film, affecting the dehydration of the internal material, and during dehydration, the tissue could not be completely dehydrated, affecting the subsequent transparent and embedding processes, and further affecting the section examination result of the tissue.
In comparative example 4, the fresh tissue of the mammary gland is fixed by using the pentanediol solution with the mass percentage of 2.5%, and as the permeability of the pentanediol is poor and the speed of fixing the tissue is slow, substances in the tissue are difficult to be completely fixed, so that the structural definition and the hierarchical definition of the tissue are poor, and in addition, in comparative example 2, the dehydration function of ethanol is not provided, and after the fixation treatment, a complete dehydration process is required, so that the consumed time is long.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A formaldehyde-free fixing solution for fixing various fresh tissues is characterized by comprising a solution A and a solution B;
the liquid A comprises picric acid and glacial acetic acid;
the liquid B comprises the following raw materials: glutaraldehyde and ethanol.
2. The formaldehyde-free fixative solution for fixation of fresh tissues of claim 1, wherein the solution B comprises solution B1, solution B2, solution B3;
the B1 liquid is prepared from the following raw materials in parts by mass: 10 parts of glutaraldehyde, 50 parts of ethanol and 40 parts of phosphate buffer;
the B2 liquid is prepared from the following raw materials in parts by mass: 10 parts of glutaraldehyde, 70 parts of ethanol and 20 parts of phosphate buffer;
the B3 liquid is prepared from the following raw materials in parts by mass: 5 parts of glutaraldehyde and 95 parts of ethanol.
3. The formaldehyde-free fixative solution of claim 1, wherein the picric acid is a saturated picric acid solution, and the mass percentage of the saturated picric acid solution to the glacial acetic acid is 15: 1.
4. a method for preparing the formaldehyde-free fixative solution of any one of claims 1-3 for fixation of multiple fresh tissues comprising the steps of:
step one, preparing solution A:
taking picric acid and glacial acetic acid according to mass percent, and stirring and mixing uniformly to obtain solution A;
step two, preparing liquid B:
preparing a B1 solution: taking glutaraldehyde, ethanol and a phosphate buffer solution according to the parts by weight, and uniformly stirring and mixing to obtain a B1 solution;
preparing a B2 solution: taking glutaraldehyde, ethanol and a phosphate buffer solution according to the parts by weight, and uniformly stirring and mixing to obtain a B2 solution;
preparing a B3 solution: taking glutaraldehyde and ethanol according to the parts by weight, stirring and mixing uniformly to prepare B3 liquid.
5. A method of fixating fresh tissue using a fixation fluid according to any of claims 1-3, comprising the steps of:
s1, placing fresh tissues into solution A for first fixation;
s2, after the first fixation is finished, washing tissues by using PBS buffer solution;
and S3, placing the cleaned tissue in a fixing chamber, adding the solution B, and performing second fixing.
6. The method of fixing fresh tissue according to claim 5,
the step of adding liquid B includes the replacement of part of the used liquid.
7. The method of fixing fresh tissue according to claim 6,
the sequence of adding each solution in the solution B in the S3 is as follows: adding the B1 solution, then adding the B2 solution and finally adding the B3 solution.
8. The method for fixing fresh tissue according to claim 7, wherein the second fixing in S3 comprises the following three liquid changing modes in sequence:
a first liquid changing mode, when B2 liquid needs to be added into the fixed chamber, if the liquid in the current fixed chamber is used B1 liquid, part of the liquid in the current fixed chamber is discharged, and B2 liquid is added to carry out first liquid changing;
a second liquid changing mode, when B2 liquid needs to be added into the fixed chamber, if the liquid in the current fixed chamber comprises the used B2 liquid, part of the liquid in the current fixed chamber is discharged, and B2 liquid is added to carry out second liquid changing;
in the third liquid changing mode, when B3 liquid needs to be added into the fixed chamber, part of the liquid in the fixed chamber is discharged and B3 liquid is added, and the third liquid changing is carried out.
9. The method of claim 6, wherein a linkage chamber is provided above the fixed chamber, a movable piston is provided between the linkage chamber and the fixed chamber, the movable piston can move up and down between the linkage chamber and the fixed chamber in a sealing manner when closed, and the movable piston moves up and down to change the volume of the fixed chamber and the linkage chamber;
the replacement of the partially used liquid comprises the following steps:
when it is necessary to replace a part of the liquid in the holding chamber,
s101, filling new liquid into the linkage chamber, descending the movable piston in a force accumulation mode, driving the linkage assembly to move to the opening of the liquid discharge hole, and discharging part of the used liquid in the fixed chamber from the liquid discharge hole in an opening state;
s102, after the piston descends to a set position along with the movement, the linkage assembly is driven to move until the liquid discharge hole is closed again;
s103, the movable piston is opened, so that the empty chamber of the movable piston is exposed, the fixed chamber, the linkage chamber and the empty chamber are communicated, new liquid is poured into the empty chamber, the stored force starts to be released, the liquid discharge hole is kept closed while the movable piston moves in a resetting mode until the movable piston is reset, the movable piston is closed, the linkage chamber and the fixed chamber are sealed and separated again, and replacement of part of used liquid is completed.
10. The method of securing fresh tissue according to claim 9, wherein the linkage assembly comprises linkage bar teeth, drive ratchet teeth; the upper end of the linkage bar-shaped tooth is rotationally matched with the movable piston; the linkage strip-shaped teeth are in one-way linkage with the transmission ratchets;
the linkage bar-shaped tooth up-and-down reciprocating motion comprises the following steps:
step one, when the linkage bar-shaped teeth move downwards, the linkage bar-shaped teeth are contacted with the transmission ratchet teeth to drive the transmission ratchet teeth to rotate in a force storage mode so as to open the liquid discharge hole;
step two, when the linkage bar-shaped teeth continue to move downwards to be separated from the transmission ratchet, the transmission ratchet releases the stored force to reset and rotate, and the liquid discharge hole is closed;
and step three, in the process of upward resetting of the linkage strip-shaped teeth, the reverse rotation of the transmission ratchets is limited due to unidirectional blocking, and the linkage strip-shaped teeth overcome the blocking of the transmission ratchets in a deflection mode and reset upward.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8614120D0 (en) * | 1986-06-10 | 1986-07-16 | Lab Supplies & Instr Antrim Lt | Histology fixative |
| CN1587959A (en) * | 2004-09-23 | 2005-03-02 | 上海交通大学 | Method for producing biolgoical sample embedded block based on atomic force microscope observation |
| CN102147337A (en) * | 2011-01-13 | 2011-08-10 | 中国水产科学研究院东海水产研究所 | Paraffin sectioning method for ocean shellfish D-shaped larva |
| CN102175496A (en) * | 2011-01-21 | 2011-09-07 | 哈尔滨格林标本技术开发有限公司 | Formaldehyde-free fixative for tissue sample |
| CN103471903A (en) * | 2013-09-29 | 2013-12-25 | 吴天骥 | Method for preparing stationary liquid for slicing and application of stationary liquid for slicing |
| CN109418258A (en) * | 2017-08-24 | 2019-03-05 | 武汉宏强医疗器械有限公司 | A kind of environmental protection fixer |
| CN109781492A (en) * | 2018-12-28 | 2019-05-21 | 广州维格斯生物科技有限公司 | It is a kind of to colour lasting tissue fixative solution |
| CN110073194A (en) * | 2016-12-22 | 2019-07-30 | 文塔纳医疗系统公司 | System and method for Sample preservation |
| US20200041389A1 (en) * | 2015-11-13 | 2020-02-06 | The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services | Fixatives and methods of use |
| CN111060374A (en) * | 2019-12-31 | 2020-04-24 | 宁夏大学 | GN aldehyde-free rapid fixing liquid and preparation method thereof |
| CN112816015A (en) * | 2019-11-15 | 2021-05-18 | 西安北斗星数码信息股份有限公司 | Water meter with automatic cover opening function |
-
2021
- 2021-12-24 CN CN202111604986.XA patent/CN114279801B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8614120D0 (en) * | 1986-06-10 | 1986-07-16 | Lab Supplies & Instr Antrim Lt | Histology fixative |
| CN1587959A (en) * | 2004-09-23 | 2005-03-02 | 上海交通大学 | Method for producing biolgoical sample embedded block based on atomic force microscope observation |
| CN102147337A (en) * | 2011-01-13 | 2011-08-10 | 中国水产科学研究院东海水产研究所 | Paraffin sectioning method for ocean shellfish D-shaped larva |
| CN102175496A (en) * | 2011-01-21 | 2011-09-07 | 哈尔滨格林标本技术开发有限公司 | Formaldehyde-free fixative for tissue sample |
| CN103471903A (en) * | 2013-09-29 | 2013-12-25 | 吴天骥 | Method for preparing stationary liquid for slicing and application of stationary liquid for slicing |
| US20200041389A1 (en) * | 2015-11-13 | 2020-02-06 | The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services | Fixatives and methods of use |
| CN110073194A (en) * | 2016-12-22 | 2019-07-30 | 文塔纳医疗系统公司 | System and method for Sample preservation |
| CN109418258A (en) * | 2017-08-24 | 2019-03-05 | 武汉宏强医疗器械有限公司 | A kind of environmental protection fixer |
| CN109781492A (en) * | 2018-12-28 | 2019-05-21 | 广州维格斯生物科技有限公司 | It is a kind of to colour lasting tissue fixative solution |
| CN112816015A (en) * | 2019-11-15 | 2021-05-18 | 西安北斗星数码信息股份有限公司 | Water meter with automatic cover opening function |
| CN111060374A (en) * | 2019-12-31 | 2020-04-24 | 宁夏大学 | GN aldehyde-free rapid fixing liquid and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 刘艳;周杰;黄宜兵;张桂荣;姜丹;邵妍;李臣亮;孟庆繁;: "组织学石蜡切片制作中固定方法的改良", 生物学通报, no. 01, pages 47 - 48 * |
| 孙河龙: "组织固定液与固定方法选择的探讨", 甘肃医药, vol. 38, no. 12, pages 1061 - 1064 * |
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