CN111982626B - Method for identifying quick paraffin section of cartilage ball - Google Patents
Method for identifying quick paraffin section of cartilage ball Download PDFInfo
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Classifications
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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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- 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
- G01N1/34—Purifying; Cleaning
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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
- G01N1/36—Embedding or analogous mounting of samples
-
- 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
- G01N1/44—Sample treatment involving radiation, e.g. heat
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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
- G01N2001/302—Stain compositions
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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
- G01N2001/305—Fixative compositions
- G01N2001/307—Fixative compositions non-toxic, no Hg, no formaldehyde
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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
- G01N1/36—Embedding or analogous mounting of samples
- G01N2001/364—Embedding or analogous mounting of samples using resins, epoxy
Abstract
The invention discloses a method for identifying a quick paraffin section of a cartilage ball, which belongs to the technical field of cell tissue identification and has the technical scheme that the method comprises the following steps of S1, preheating water: preheating water to the water temperature of 60 ℃ by adopting microwave heating; s2, tissue treatment: transferring the tissue to a clean petri dish; s3, fixing tissues; s4, dehydrating tissues; s5, completely dehydrating: transferring the tissue into acetone, and heating and dehydrating by microwaves; s6, tissue transparent treatment; s7, tissue waxing treatment: transferring the tissue into a wax block I and a wax block II in turn for microwave heating; s8, preparing tissue paraffin sections; s9, judging a tissue result: the invention has the advantages of shortening the identification time, controlling the molecular high-frequency vibration to obviously accelerate the steps of fixation, dehydration and the like when a microscope is used for observing tissues, keeping the tissue structure and the cell morphology completely, and improving the progress of experiments and the quality of results.
Description
Technical Field
The invention relates to the technical field of cell tissue identification, in particular to a method for identifying a quick paraffin section of a cartilage ball.
Background
The traditional pathological paraffin section basic steps comprise the steps of tissue fixation, dehydration, transparency, wax dipping, embedding, section, dyeing and the like, wherein each step is basically at least 1 hour, the whole course is at least 16 hours, and if the pathological paraffin section is used in winter, the operation time is longer due to lower room temperature. Dehydration and transparency are the most critical steps in slicing, dehydration is performed for nearly six hours completely, and xylene transparency is performed for nearly 1 hour. If the tissue is not completely dehydrated, the tissue appears as a mist and the procedure needs to be repeated appropriately.
Induction of umbilical cord stem cells into intact chondrocytes takes about 20 days, and the spheroid volume of cartilage is tiny, about 0.1 x 0.1cm. If the traditional paraffin section is used for judging the induction experimental result, the experimental process is long, the dehydration transparent process is not easy to control, and the tissue dehydration is easy to be insufficient, so that the section quality is affected. Therefore, the conventional paraffin section technique is not well suited for morphological identification of the results of such stem cell induced differentiation experiments.
Disclosure of Invention
The invention aims to provide a rapid paraffin section identification method for cartilage balls, which has the advantages of shortening the identification time, obviously accelerating the steps of fixation, dehydration and the like by controlling molecular high-frequency vibration, keeping the tissue structure and the cell morphology completely, and improving the progress and the result quality of experiments.
The technical aim of the invention is realized by the following technical scheme:
a method for identifying a cartilage ball rapid paraffin section comprises the following steps:
s1, preheating water: preheating water to water temperature of 60 ℃ by adopting microwave heating, and melting the wax blocks I and II by using a constant-temperature drying oven;
s2, tissue treatment: transferring the tissue to a clean culture dish, and washing off the surface culture medium with clear water;
s3, fixing tissues: placing the tissue into a test tube, and then adding a rapid fixing liquid into the test tube for microwave heating;
s4, dehydration of tissues: transferring the tissue in the test tube into ethanol with the concentration of 60%, 70%, 80% and 90% in sequence for dehydration;
s5, completely dehydrating: transferring the tissue into acetone, and heating and dehydrating by microwaves;
s6, tissue transparent treatment: sequentially transferring the tissues into a test tube with xylene I and a test tube with xylene II for microwave heating respectively;
s7, tissue waxing treatment: firstly, melting a wax block I (52 ℃) and a wax block II (55 ℃) in a constant-temperature drying oven, and then sequentially transferring tissues into the wax block I and the wax block II for microwave heating;
s8, preparing tissue paraffin sections: embedding the tissues by using a wax block II, then putting the embedded tissues into a slicing machine to cut out required wax sheets, putting the wax sheets into water for bleaching, then using a glass slide patch with an adhesive to patch, roasting the wax sheets after the patch is finished, finally slicing, dewaxing and rehydrating, and then dyeing;
s9, judging a tissue result: the tissue was observed using a microscope.
Further, in step S1, 150ml of water was added using a 200ml beaker, and the water was heated for 1min, and wax block I (52 degrees) and wax block II (55 degrees) were melted using a beaker having a volume of 100 ml.
Further, in step S3, the rapid fixative solution formulation is: ethanol, diethyl ether, acetic acid, polyethylene glycol-40, glycerol, salicylic acid, EDTA and xylose, wherein the concentrations of the components are 20% of ethanol, 20% of diethyl ether, 30% of acetic acid, 20% of polyethylene glycol-400.10% of glycerol, 0.5mol/L of EDTA, 0.5mg/ml of xylose and 3.6g/ml of salicylic acid.
Further, in step S3, the temperature range of the microwave heating is 30-40 ℃ and the heating time is 3min.
Further, in step S4, 6ml of ethanol is used for each dehydration, and each dehydration is heated by microwave heating for 3min at 30-40deg.C.
Further, in step S5, acetone was used at 6ml, and the heating was performed for 3min with microwaves at a temperature ranging from 30 to 40 ℃.
Further, in step S6, the microwave heating is performed for 3min at a heating temperature ranging from 30 to 40 ℃.
Further, in step S7, in the wax block I and the wax block II, the heating is performed for 3min at a temperature ranging from 50 to 60 ℃.
Further, in step S8, the temperature range of the baked chips is 30-40 ℃ and the time for baking the chips is 3min.
Further, in the dyeing process of the step S8, the slice is dyed by using an aliskirin blue dye solution, then the slice is incubated for 30min at normal temperature, the slice is washed for 2min by using clean water, the slice is washed for 2min by using pure water, the slice is dehydrated, and finally the neutral resin is used for sealing the slice.
In summary, the invention has the following beneficial effects:
1. in the whole experimental process, the microwave radiation technology is used, the steps of fixing, dehydrating and the like are obviously accelerated by high-frequency vibration of molecules under a magnetic field, meanwhile, the tissue structure and the cell morphology can be preserved completely, and meanwhile, the water bath method is used in a combined mode, because water can absorb the microwave radiation most quickly and effectively, the solution in the test tube can be heated uniformly, and the stability and the replicability of the experiment are ensured.
2. The traditional fixing solution is 4% paraformaldehyde, the paraformaldehyde has mild effect, but can harden tissues, the tissues are easy to become brittle during fixing and fragile during slicing, the paraformaldehyde can exist in fixed cells or tissue samples for a long time, residual aldehyde groups still exist after the fixing is finished after the fixing is washed for a plurality of hours by using washing solution or water, the residual aldehyde groups can influence the subsequent experimental results, in addition, the paraformaldehyde is harmful to human bodies, and in the operation process, the paraformaldehyde is difficult to avoid contacting the human bodies or being inhaled into the human bodies. Based on the above reasons, we select a rapid fixing solution for fixing, wherein the components of the rapid fixing solution comprise ethanol, diethyl ether, salicylic acid, acetic acid, polyethylene glycol-400, glycerol, EDTA and xylose, and the salicylic acid has the effect of softening cartilage to dissolve fat so as to dredge the tissue gap. Heating may use thermal coagulation of proteins, but may bring about the side effects of stiffening and shrinking the specimen, and salicylic acid may combat this undesirable factor. Acetic acid counteracts the contraction effect of ethanol on tissues by expanding tissue fibers, diethyl ether enhances the denaturation effect of ethanol on proteins, polyethylene glycol enhances the penetration effect of a fixing agent on cell membranes, glycerol is also called glycerol, the refractive index of tissues can be changed, the dyed color is vivid, EDTA is taken as a protease inhibitor, xylose is also called ribulose, and the xylose is an aldopentose, can protect the integrity of tissue proteins and DNA, is safe and nontoxic, has strong operability, can better and more completely retain the original tissue results of cartilage balls, and ensures more reliable experimental results and clearer experimental pictures.
3. In the dehydration process, acetone is used for replacing the dehydration effect of 95% ethanol and 100% ethanol, so that the dehydration effect is more thorough and rapid, and the phenomenon of tissue fogging in the transparent process is reduced.
Drawings
FIG. 1 is a schematic flow chart of a method for identifying a rapid paraffin section of a cartilage ball;
FIG. 2 is a photograph of cartilage ball at 200 Xmagnification;
fig. 3 is a photograph of cartilage ball at 400X magnification.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
Examples: a method for identifying a quick paraffin section of a cartilage ball is shown in figure 1, and comprises the following steps:
s1, preheating water: about 150ml of water is added into a 200ml beaker, the water is heated for 1min by adopting a microwave heating mode, the heating temperature range is 70-80 ℃, and finally the water is preheated to 60 ℃. A thermostatic oven was prepared and set to 60 ℃, wax block I and wax block II were melted in a 100ml beaker.
S2, tissue treatment: the tissue was transferred to a clean petri dish and the surface medium was washed off with clean water.
S3, fixing tissues: firstly, preparing a quick fixing liquid, wherein the formula is as follows: ethanol, diethyl ether, acetic acid, polyethylene glycol-40, glycerol, salicylic acid, EDTA and xylose. The concentration of each component in the rapid fixing solution is as follows: 20% of ethanol, 20% of diethyl ether, 30% of acetic acid, 400.10% of polyethylene glycol, 20% of glycerol, 0.5mol/L of EDTA0.5mol/L of xylose, 0.5mg/ml of salicylic acid and 3.6g/ml of salicylic acid.
The preparation method of the quick fixing liquid comprises the following steps:
(1) Mixing 20ml of ethanol, 10ml of polyethylene glycol-400 and 20ml of glycerol, stirring uniformly, and placing in a refrigerator at 4 ℃ as A solution for later use.
(2) Mixing 20ml of diethyl ether and 30ml of acetic acid, stirring uniformly, and placing in a refrigerator at 4 ℃ as liquid B for later use.
(3) And uniformly mixing the solution A and the solution B, then dropwise adding and stirring, adding 0.05mol of EDTA and 50mg of xylose until the solution is clear and transparent, and finally adding 36g of salicylic acid to adjust the pH range of the quick fixing solution to 7.1-7.3.
After the preparation of the quick fixing liquid is completed, the tissue is placed into a medium-sized test tube, 6ml of the quick fixing liquid is added into the medium-sized test tube, the medium-sized test tube is placed into a beaker, water is added into the beaker, and the tissue is heated for 3min in a microwave heating mode, wherein the heating temperature is 30-40 ℃.
The traditional fixing solution is 4% paraformaldehyde, the paraformaldehyde has mild effect, but can harden tissues, the tissues are easy to become brittle during fixing and fragile during slicing, the paraformaldehyde can exist in fixed cells or tissue samples for a long time, residual aldehyde groups still exist after the fixing is finished after the fixing is washed for a plurality of hours by using washing solution or water, the residual aldehyde groups can influence the subsequent experimental results, in addition, the paraformaldehyde is harmful to human bodies, and in the operation process, the paraformaldehyde is difficult to avoid contacting the human bodies or being inhaled into the human bodies. Based on the above reasons, we select a rapid fixing solution for fixing, wherein the components of the rapid fixing solution comprise ethanol, diethyl ether, salicylic acid, acetic acid, polyethylene glycol-400, glycerol, EDTA and xylose, and the salicylic acid has the effect of softening cartilage to dissolve fat so as to dredge the tissue gap. Heating may use thermal coagulation of proteins, but may bring about the side effects of stiffening and shrinking the specimen, and salicylic acid may combat this undesirable factor. Acetic acid counteracts the contraction effect of ethanol on tissues by expanding tissue fibers, diethyl ether enhances the denaturation effect of ethanol on proteins, polyethylene glycol enhances the penetration effect of a fixing agent on cell membranes, glycerol is also called glycerol, can change the refractive index of tissues, so that the dyed color is vivid, EDTA is used as a protease inhibitor, and the chemical formula is C 10 H 16 N 2 O 8 Can be combined with Mg 2+ Divalent metal ion binding due to Mg 2+ The EDTA is an ion required by the action of proteases, so that the EDTA can inhibit the activity of the proteases, protect the protein structure, prevent the protein from being degraded and ensure the integrity of tissues. Xylose, also called ribulose, is an aldopentose, which can protect the integrity of DNA, the fixing liquid is safe and nontoxic, has strong operability, and can better and completely retain the original tissue result of cartilage balls, so that the experimental result is more reliable and the experimental picture is clearer.
S4, dehydration of tissues: the tissues in the test tube were sequentially transferred to 60%, 70%, 80% and 90% ethanol for dehydration, and the amount of ethanol used was 6ml for each group. Each dehydration is heated by microwave heating for 3min at 30-40deg.C.
S5, completely dehydrating: the tissue was placed in a medium test tube with acetone, which was taken in an amount of 6ml. And then heating for 3min by using a microwave heating mode, wherein the heating temperature is 30-40 ℃. The method has the advantages that the acetone is used for replacing the dehydration effect of 95% ethanol and 100% ethanol in the complete dehydration process, so that the dehydration effect is more thorough and rapid, and the phenomenon of tissue fogging in the transparent process is reduced.
S6, tissue transparent treatment: the tissue was sequentially transferred to a medium test tube containing xylene I and a medium test tube containing xylene II, each of which was taken in an amount of 6ml, for microwave heating. The conditions of microwave heating are: heating by microwave for 3min at 30-40deg.C.
S7, tissue waxing treatment: melting wax block I (52 deg.C) and wax block II (55 deg.C) in a constant temperature drying oven, sequentially transferring the tissues into the wax block I and the wax block II, and respectively heating with microwave for 3min at 50-60deg.C.
S8, preparing tissue paraffin sections: the preparation method comprises the steps of embedding tissues by using redundant wax blocks II, cutting out needed wax sheets in a slicing machine, then placing the wax sheets into water with the temperature of 42 ℃ for bleaching, then attaching a glass slide with an adhesive, baking the sheets for 3min in a microwave heating mode after the attaching is finished, wherein the temperature range of the baked sheets is 30-40 ℃, finally carrying out dewaxing and rehydration steps, and then dyeing by using alismatine blue dye liquor. After dyeing, incubating the wax flakes for 30min at normal temperature, washing the wax flakes with clean water for 2min, washing the wax flakes with pure water for 2min, dehydrating the wax flakes, and finally sealing the flakes with neutral resin.
S9, judging a tissue result: the tissue slice is dewaxed and rehydrated, is dyed with aliskirin blue dye liquor at normal temperature for half an hour, is washed twice with clear water, is dehydrated in a gradient way for 2 minutes each time, is sealed with neutral resin, and is observed under a microscope.
And (3) tissue structure display: cartilage balls at 200X magnification are shown in figure 2; cartilage balls are shown in figure 3 at 400X magnification.
Conclusion: the alisxin blue staining part shows that the internal acid mucopolysaccharide in the cartilage ball has good slice quality, more complete morphology and clearer tissue structure compared with the conventional paraffin slice.
The present embodiment is only for explanation of the present invention and is not to be construed as limiting the present invention, and modifications to the present embodiment, which may not creatively contribute to the present invention as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present invention.
Claims (9)
1. A method for identifying a quick paraffin section of a cartilage ball is characterized by comprising the following steps: the method comprises the following steps:
s1, preheating water: preheating water to water temperature of 60 ℃ by adopting microwave heating, and melting the wax blocks I and II by using a constant-temperature drying oven;
s2, tissue treatment: transferring the tissue to a clean culture dish, and washing off the surface culture medium with clear water;
s3, fixing tissues: placing the tissue into a test tube, and then adding a rapid fixing liquid into the test tube for microwave heating; firstly, preparing a quick fixing liquid, wherein the formula is as follows: the concentration of each component in the quick fixing solution is as follows: 20% of ethanol, 20% of diethyl ether, 30% of acetic acid, 400.10% of polyethylene glycol, 20% of glycerol, 0.5mol/L of EDTA, 0.5mg/ml of xylose and 3.6g/ml of salicylic acid;
the preparation method of the quick fixing liquid comprises the following steps:
(1) Mixing 20ml of ethanol, 10ml of polyethylene glycol-400 and 20ml of glycerol, uniformly stirring, and placing the mixture in a refrigerator at 4 ℃ as A solution for later use;
(2) Mixing 20ml of diethyl ether and 30ml of acetic acid, stirring uniformly, and placing the mixture as liquid B in a refrigerator at 4 ℃ for later use;
(3) Uniformly mixing the solution A and the solution B, then dropwise adding and stirring, adding 0.05mol of EDTA and 50mg of xylose until the solution is clear and transparent, and finally adding 36g of salicylic acid to adjust the pH range of the quick fixing solution to 7.1-7.3;
s4, dehydration of tissues: transferring the tissue in the test tube into ethanol with the concentration of 60%, 70%, 80% and 90% in sequence for dehydration;
s5, completely dehydrating: transferring the tissue into acetone, and heating and dehydrating by microwaves;
s6, tissue transparent treatment: sequentially transferring the tissues into a test tube with xylene I and a test tube with xylene II for microwave heating respectively;
s7, tissue waxing treatment: firstly, melting a wax block I and a wax block II in a constant-temperature drying oven, wherein the melting point of the wax block I is 52 DEG, the melting point of the wax block II is 55 DEG, and then sequentially transferring tissues into the wax block I and the wax block II for microwave heating;
s8, preparing tissue paraffin sections: embedding the tissues by using a wax block II, then putting the embedded tissues into a slicing machine to cut out required wax sheets, putting the wax sheets into water for bleaching, then using a glass slide patch with an adhesive to patch, roasting the wax sheets after the patch is finished, finally slicing, dewaxing and rehydrating, and then dyeing;
s9, judging a tissue result: the tissue was observed using a microscope.
2. The method for identifying the rapid paraffin section of the cartilage ball according to claim 1, wherein the method comprises the following steps: in step S1, 150ml of water is added by using a 200ml beaker, the water is heated for 1min, a beaker with the volume of 100ml is selected to melt the wax block I and the wax block II, the melting point of the wax block I is 52 degrees, and the melting point of the wax block II is 55 degrees.
3. The method for identifying the rapid paraffin section of the cartilage ball according to claim 1, wherein the method comprises the following steps: in step S3, the temperature range of microwave heating is 30-40 ℃ and the heating time is 3min.
4. The method for identifying the rapid paraffin section of the cartilage ball according to claim 1, wherein the method comprises the following steps: in step S4, 6ml of ethanol is used for each dehydration, and each dehydration is heated by microwave heating for 3min at 30-40deg.C.
5. The method for identifying the rapid paraffin section of the cartilage ball according to claim 1, wherein the method comprises the following steps: in step S5, 6ml of acetone was used and heated by microwaves for 3min at a temperature ranging from 30 to 40 ℃.
6. The method for identifying the rapid paraffin section of the cartilage ball according to claim 1, wherein the method comprises the following steps: in step S6, the microwave heating is performed for 3min at a temperature ranging from 30 to 40 ℃.
7. The method for identifying the rapid paraffin section of the cartilage ball according to claim 1, wherein the method comprises the following steps: in step S7, in the wax block I and the wax block II, the heating is performed for 3min at a temperature ranging from 50 to 60 ℃.
8. The method for identifying the rapid paraffin section of the cartilage ball according to claim 1, wherein the method comprises the following steps: in the step S8, the temperature range of the baked chips is 30-40 ℃ and the time for baking the chips is 3min.
9. The method for identifying the rapid paraffin section of the cartilage ball according to claim 1, wherein the method comprises the following steps: in the dyeing process of the step S8, the wax sheet is dyed by using an aliskirin blue dye solution, then the wax sheet is incubated for 30min at normal temperature, the wax sheet is washed for 2min by using clear water, the wax sheet is washed for 2min by using pure water, the wax sheet is dehydrated, and finally the neutral resin is used for sealing the sheet.
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