Method for fixing muscular tissue suitable for transportation, and method for fluorescent staining of muscular tissue

Abstract

The invention belongs to the technical field of biology, and discloses a muscular tissue fixing method suitable for transportation, which comprises the following steps: placing a freshly obtained muscle sample in a formalin solution with the concentration of 8-12wt%, and fixing for 14-18h; and then, immersing the fixed muscle sample in an ethanol solution with the concentration of 60-80 vol% for preservation. According to the method, the tissue sample is fixed by formalin and soaked in an ethanol solution, so that the tissue sample can be stored, preserved and transported for a long time, the subsequent fluorescent staining is not influenced, and in the experiment process, the subsequent fluorescent staining is not influenced even if the tissue sample is preserved for half a year by adopting the method disclosed by the invention. Meanwhile, the invention also provides a fluorescent staining method of the muscle tissue.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for fixing muscle tissue suitable for transportation and a method for fluorescent staining of muscle tissue. Background Art

[0002] Immunofluorescence histochemistry, as an effective means of detecting and locating antigenic substances in tissues, has been widely used in the fields of medicine, botany and zoology. At present, this technology can achieve immunofluorescence staining through paraffin sections and frozen sections. When paraffin sections are used for immunofluorescence histochemistry, they need to go through complex steps such as dehydration, transparency, wax immersion during section preparation, and dewaxing during staining. When this technology uses traditional frozen sections, liquid nitrogen is required for quick freezing, and liquid nitrogen is dangerous during transportation; during the liquid nitrogen quick freezing process, the tissue sample will crack after being frozen in liquid nitrogen for a long time. At the same time, the water in the tissue sample will form ice crystals due to the quick freezing process, and the ice crystals formed will destroy the morphological structure of the tissue during the later staining process; dry ice or liquid nitrogen is required for sample transportation, and the cost of liquid nitrogen and dry ice is high; in addition, the sample needs to be stored in an ultra-low temperature refrigerator, and the sampling process will also be subject to geographical restrictions.

[0003] Therefore, in the existing field of immunofluorescence histochemistry, it is generally necessary to use on-site sampling and on-site freezing methods to preserve samples; For details, please refer to the following literature: Prior art 1: A patent application with publication number CN114058667A, which is a method for verifying the effect of anticancer drugs on colon cancer cells, states: Step D of constructing and testing a nude mouse xenograft tumor model includes modeling and drug administration, detection indicators and calculation procedures; in frozen section detection, the removed tumor tissue is immersed in formalin for 2-3 days, then the tumor tissue is embedded with an OTC embedding agent, and sliced ​​on a freezing microtome, and the obtained frozen sections are used for subsequent EdU staining.

[0004] This method is used to slice tumor tissue, which requires long-term immersion in formalin and then OTC embedding. In the on-site processing of bovine muscle tissue, it has been verified that it cannot guarantee that muscle samples can be preserved for a long time.

[0005] Prior art 2: Publication number CN116893172A, the subject is an automatically aligned tumor clinical treatment slice observation device and its use method, which records: S1, the tissue specimen is fixed with 10% neutral formalin buffer fixative within 30 minutes after ex vivo, then the tissue specimen is sliced ​​and placed in an embedding box, and soaked in ethanol solution for 7 hours, then transparentized with xylene I and xylene II for 30 minutes each, and immersed in wax at 65°C for 3 hours, then the dehydrated, transparent and waxed tissue is made into a wax block, the wax block is placed on a tissue slicer to cut out 4-5μm thin slices mounted on a blank glass slide, and the slices are baked for about 30 minutes, and finally the slices are stained with HE and sealed with neutral gum, and the tissue slices are now complete.

[0006] This method requires that the tumor tissue be fixed and then immediately sliced ​​and immersed in ethanol and xylene. The purpose of these two steps is to dehydrate and transparentize the tissue, which is a pre-treatment step before wax immersion.

[0007] It can be found from the above literature that the above experimental conditions are mostly very sufficient.

[0008] However, for large animal farms, it is unrealistic to obtain perfect samples and conduct tests immediately, as the conditions for building a laboratory are not available on site.

[0009] At the same time, in the process of fixing and staining some cancer cell samples, formalin is also used for fixation, but the detected proteins are mostly single proteins. Taking EGFr as an example, EGFr can be stained normally in the immune recognition process without antigen repair (AR) using this method. If antigen repair is performed, the immune staining disappears completely. This shows that the influencing factors of fluorescent staining of muscle tissue and fluorescent staining of cancer cell samples are completely different. At the same time, the traditional method only detects a single protein when detecting cancer cells. In the requirements of the present invention, when fluorescent labeling of type I and IIa myosin, type IIa and IIx myosin is performed on the same muscle sample, it is more difficult and the influencing factors are more complicated.

[0010] Therefore, the core difficulty and demand of this case lies in how to achieve long-term storage and transportation of tissue samples under non-cryogenic conditions without affecting subsequent multiple fluorescent staining. Summary of the invention

[0011] The purpose of the present invention is to provide a method for fixing muscle tissue suitable for transportation. The present invention uses formalin to fix tissue samples and immerse them in ethanol solution. They can be stored, preserved and transported for a long time without affecting subsequent fluorescent staining. During the experiment, we found that using the method of the present invention, even if it is stored for half a year, it will not affect subsequent fluorescent staining.

[0012] At the same time, the invention also provides a method for fluorescent staining of muscle tissue.

[0013] To achieve the above object, the present invention provides the following technical solutions: A method for fixing muscle tissue suitable for transportation, wherein a freshly obtained muscle sample is placed in a formalin solution with a concentration of 8-12wt% for fixation for 14-18h; then, the fixed muscle sample is immersed in an ethanol solution with a concentration of 60-80vol% for preservation.

[0014] During the experiment, the present invention tested different formalin immersion times and different immersion solutions. During the long immersion process, the method of the present invention can extend the storage time to at least half a year, which can give sampling personnel enough time to transport samples and conduct centralized testing in the laboratory.

[0015] In the above-mentioned method for fixing muscle tissue for transportation, the freshly obtained muscle sample is placed in a formalin solution with a concentration of 10 wt % for fixation for 16 h; then, the fixed muscle sample is immersed in an ethanol solution with a concentration of 70 vol % for preservation.

[0016] In the above-mentioned method of fixing muscle tissue for transportation, the storage temperature is 4°C.

[0017] In the above-mentioned method for fixing muscle tissue for transportation, the size of the muscle sample is: 0.8-1.2 cm in length, 0.8-1.2 cm in width, and 0.3-1.0 cm in height.

[0018] At the same time, the present invention also discloses a method for fluorescent staining of muscle tissue, wherein the muscle sample is first fixed by any of the above methods; and then the fixed muscle sample is sent to a laboratory for sectioning and fluorescent staining.

[0019] In the above method, the slicing method is: The muscle samples were removed from the ethanol solution and dehydrated; The dehydrated muscle samples were then embedded and frozen using OTC embedding medium and sectioned.

[0020] In the above method, the fluorescent staining method is: The tissue sections after sectioning are fixed, antigen repaired, blocked, first incubated with primary antibody, first incubated with secondary antibody, first incubated with dye, blocked, second incubated with primary antibody, second incubated with secondary antibody, second incubated with dye, and then sealed and tested.

[0021] In the above method, the antigen repair agent used in the antigen repair is EDTA, and the specific operation of antigen repair is: placing the fixed tissue section in EDTA antigen repair solution, heating it by microwave at low heat for 10 minutes, and naturally cooling it to room temperature.

[0022] The best antigen repair method suitable for the above-mentioned sample fixation method is the EDTA microwave antigen repair method. In the screening of antigen repair methods, the present invention verified the sodium citrate boiling water heating repair method, the sodium citrate microwave heating repair method, the EDTA microwave heating repair method, and the EDTA high-pressure heating repair method. After repeated verification, the repair method most suitable for the fixation method of the present invention is to repair with EDTA antigen repair solution in a microwave oven at low heat for 10 minutes, and the positive muscle fiber morphology is brighter and clearer.

[0023] In the above method, the primary antibody used in the first incubation is BF-35 primary antibody; the secondary antibody used in the first incubation is HRP secondary antibody; the primary antibody used in the second incubation is SC-71 primary antibody; the secondary antibody used in the second incubation is HRP secondary antibody; The dye used in the first incubation was TSA-if488 staining working solution; the dye used in the second incubation was TSA-CY3 staining working solution.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses formalin to fix tissue samples and immerse them in ethanol solution, which can be stored, preserved and transported for a long time without affecting subsequent fluorescent staining. During the experiment, we found that using the method of the present invention, even if it is stored for half a year, it will not affect subsequent fluorescent staining. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1A This is an immunofluorescence staining effect diagram of the frozen section of the sample fixed for 8 hours in Example 1; Figure 1B This is an immunofluorescence staining effect diagram of the frozen section of the sample fixed for 16 hours in Example 1; Figure 1C This is an immunofluorescence staining effect diagram of the frozen section of the sample fixed for 32 hours in Example 1; Figure 2 is an immunofluorescence staining effect diagram of a frozen section of the sample of Example 2; Figure 3This is an immunofluorescence staining effect diagram of the frozen section of the sample of Comparative Example 1; Figure 4 This is an immunofluorescence staining effect diagram of the frozen section of the sample of Comparative Example 2; Figure 5 This is an immunofluorescence staining effect diagram of the frozen section of the sample of Comparative Example 3; Fig. 6A This is the effect diagram of using primary antibody BF-35 to detect the specific expression of type I and IIa myosin; Figure 6B This is the effect diagram of using primary antibody SC-71 to detect the specific expression of type IIa and IIx myosin; Figure 6C express Fig. 6A and Figure 6B The effect diagram after merging; Fig. 7A This is an immunofluorescence staining effect diagram of the sample of Comparative Example 4; Figure 7B This is an immunofluorescence staining effect diagram of the sample of Comparative Example 5; Figure 7C This is an immunofluorescence staining effect diagram of the sample of Comparative Example 6; Fig. 8A This is an immunofluorescence staining effect diagram of the sample of Comparative Example 7; Figure 8B This is an immunofluorescence staining effect diagram of the sample of Comparative Example 8; Fig. 9A This is a diagram showing the immunofluorescence staining effect of the sample of Comparative Example 9 after high-pressure repair with EDTA antigen repair solution for 3 minutes; Fig. 9B This is a diagram showing the immunofluorescence staining effect of the sample of Comparative Example 9 after high-pressure repair with sodium citrate antigen repair solution for 10 minutes; Fig. 10A This is a picture showing the effect of using the primary antibody BF-35 to detect the specific expression of type I and IIa myosin in the samples of the third part; Fig. 10B This is a picture showing the effect of using the primary antibody SC-71 to detect the specific expression of type IIa and IIx myosin in the samples of the third part; Fig. 10C express Fig. 10A and Fig. 10B The effect diagram after merging; Fig. 10D This is a staining photograph of a sample repaired in a boiling water bath for 10 min using EDTA antigen repair solution; Fig. 10E This is a staining photograph of a sample repaired in a boiling water bath for 10 min using sodium citrate antigen repair solution; Fig.10FThis is a staining photograph of a sample that was repaired with sodium citrate antigen repair solution at low temperature in a microwave oven for 10 minutes. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Part I. Optimization of sample fixation process

[0028] Example 1 In this experiment, the quadriceps femoris muscle of Lufeng cattle was selected, and the samples were from the slaughterhouse. Lufeng cattle were slaughtered according to the standard slaughter process. Within 30 minutes of slaughtering Lufeng cattle, the quadriceps femoris muscle was collected, the fascia was removed, and the muscles were cut into 1*1*0.5 cm in length*width*height along the direction of the muscle fibers. 3 The muscle sample was fixed in 10wt% neutral formalin (the volume of 10% neutral formalin should not be less than 20 times the volume of the tissue sample) at 4℃ for 16 hours. After fixation, the sample was transferred to a centrifuge tube containing 70vol% alcohol and transported and stored at 4℃.

[0029] The fixation time of the above neutral formalin is 8h, 16h, and 32h; The above samples were frozen and sectioned after being stored for 7 days; The operation of frozen section is as follows: take out the tissue sample from the centrifuge tube containing 70 vol% alcohol, absorb the alcohol on the surface with filter paper, and dehydrate it in a centrifuge tube containing 30 wt% sucrose solution (prepared with sterile water) twice, each time the sample sinks to the bottom of the centrifuge tube.

[0030] OTC embedding and sectioning: Use filter paper to absorb the sucrose solution on the tissue surface, embed and section the tissue sample with OTC embedding agent (biosharp) at -30℃ using a Leica cryostat, with a section thickness of 8μm. After sectioning, place the section in a 37℃ oven for 1 h before proceeding to the next step of fluorescent staining.

[0031] The operation of fluorescent staining can be referred to the specific operation in Part 2 and will not be further elaborated here.

[0032] Reference for immunofluorescence staining of frozen sections of samples fixed for 8 hours Figure 1A ; Reference for immunofluorescence staining of frozen sections of samples fixed for 16 hours Figure 1B ; Reference for immunofluorescence staining of frozen sections of samples fixed for 32 hours Figure 1C .

[0033] pass Figures 1A to 1C As shown in the figure, after fixation with 10% neutral formalin for 8 h and 32 h, the morphology of positive muscle fibers was incomplete, while the morphology of positive muscle fibers at 16 h was relatively complete.

[0034] Example 2 The same as Example 1, the fixation time is 16 hours, except that the samples are frozen and sectioned and stained after being stored for 180 days. Figure 2 .

[0035] pass Figure 2 It can be seen that the positive muscle fiber morphology is relatively complete, and Figure 1B There is no significant difference.

[0036] Comparative Example 1 The same as Example 1, the fixation time is 16h, except that the sample is stored in 60vol% ethanol solution, frozen sectioned and stained, and the photo is referenced. Figure 3 ;pass Figure 3 It can be seen that the morphology of the positive muscle fibers of the sample is not complete.

[0037] Comparative Example 2 The same as Example 1, the fixation time is 16h, except that the sample is stored in 75vol% ethanol solution, frozen sectioned and stained, and the photo is referenced. Figure 4 ;pass Figure 4 It can be seen that the morphology of the positive muscle fibers of the sample is not complete.

[0038] Comparative Example 3 The same as Example 1, except that the fixation time is 16 hours, the fixative is a paraformaldehyde solution, the paraformaldehyde solution contains 4wt% paraformaldehyde; 1.9 mM sodium dihydrogen phosphate dihydrate; 8.1 mM disodium hydrogen phosphate dodecahydrate, and the frozen sections are stained, and the photos are referenced. Figure 5 ;pass Figure 5 It can be seen that the morphology of the positive muscle fibers of the sample is not complete.

[0039] From the above cases, it can be seen that the use of appropriate fixation time, appropriate type of preservation solution, and appropriate fixation solution are the key to ensuring that the sample can be displayed in the form of complete positive muscle fibers.

[0040] Part II Optimization of Fluorescent Dyeing Process

[0041] Example 3 The sample fixed for 16 hours in Example 1 was treated as follows and then fluorescently stained.

[0042] The specific steps are: Tissue sections were rinsed in a slow stream of tap water for 5 min; (1) Fix with 10wt% formalin for 13 min and wash with sterile water 3 times, 1 min each time; (2) Antigen retrieval: Place an incubation wet box containing 200 mL of 1x EDTA antigen retrieval solution (Solabo, 50x EDTA antigen retrieval solution diluted to 1x with sterile water) in a microwave oven and boil it at high heat. Place the tissue sections in the microwave oven at low heat for 10 minutes and allow to cool naturally to room temperature. The low-fire parameters of the microwave oven are microwave power: 300w, microwave frequency: 2450 MHz; (3) Washing: Wash 3 times with PBS, 5 min each time; (4) Draw a circle: Use an immunohistochemistry pen to draw a circle around the sample; (5) Blocking: Add 100 μL of 10% goat serum (Solabo) to the sample, mix well, and incubate with shaking at room temperature for 30 min. (6) Incubation with primary antibody: remove the blocking serum with a pipette tip, add 60 μL of BF-35 primary antibody (primary antibody: diluent = 3:8.5 (volume ratio), diluted with antibody diluent, DSHB), mix well, and incubate in a refrigerator at 4°C for 24 h; (7) Take out the slices and let them stand at room temperature for 1 h; (8) Washing: Wash 3 times with PBS, 5 min each time; (9) Incubation with secondary antibody: add 60 μL of HRP secondary antibody (Absin) to the sample, mix well, and incubate with shaking at room temperature for 4 h; (10) Washing: Wash 3 times with PBS, 5 min each time; (11) Incubation with dye: add 60 μL of TSA-if488 working solution (1:500; Seville, see Table 1) to the sample, mix well, and incubate with shaking at room temperature for 30 min; (12) Washing: Wash 3 times with PBS, 5 min each time; (13) Blocking: Add 100 μL of 10 wt% goat serum (Solebol) to the sample, mix well, and incubate with shaking at room temperature for 30 min. (14) Incubation with primary antibody: remove the blocking serum with a pipette tip, add 60 μL of SC-71 primary antibody (primary antibody: diluent = 1:13 (volume ratio), diluted with antibody diluent, DSHB), mix well, and incubate in a refrigerator at 4°C for 24 h; (15) Take out the slices and let them stand at room temperature for 1 h. (16) Washing: Wash 3 times with PBS, 5 min each time; (17) Incubation with secondary antibody: add 60 μL of HRP secondary antibody (Absin) to the sample, mix well, and incubate with shaking at room temperature for 4 h; (18) Washing: Wash 3 times with PBS, 5 min each time; (19) Incubation with dye: add 60 μL of TSA-CY3 working solution (1:500; Seville, see Table 1) to the sample, mix well, and incubate with shaking at room temperature for 30 min; (20) Washing: Wash three times with PBS, 5 min each time; if multiple immunofluorescence staining is continued, steps (6) to (13) can be repeated; (21) DAPI nuclear staining: add 100 μL of DAPI solution (Sevier) to the sample, mix well, and incubate with shaking at room temperature for 10 min; (22) Washing: Wash 3 times with PBS, 5 min each time; (23) Sealing: Add a few drops of anti-fluorescence quenching agent on the sample and cover with a coverslip; (24) Microscopic examination: Fluorescence photography was performed under a fluorescence microscope (ECLIPES-U research-grade inverted fluorescence microscope, Nikon).

[0043] Table 1 TSA staining working solution system

[0044] In this example, the immunofluorescence results of frozen sections are shown in FIG. 6A to FIG. 6C . Fig. 6A A diagram showing the effect of using the primary antibody BF-35 to detect the specific expression of type I and IIa myosin; Figure 6B A diagram showing the effect of using the primary antibody SC-71 to detect the specific expression of type IIa and IIx myosin; Figure 6C express Fig. 6A and Figure 6B The merged image shows the effect of the slice thickness of 8 μm and the scale bar of 100 μm.

[0045] The results showed that the morphological structure of the muscle fibers remained intact and the fluorescent labeled proteins were clear. In the figure, green fluorescence indicated type I and IIa muscle fibers (Figure 6 A), and red fluorescence indicated type IIa and IIx muscle fibers (Figure 6 B). After the two fluorescences were combined, type I (green), type IIa (yellow), and type IIx (red) muscle fibers could be clearly distinguished (Figure 6 C).

[0046] The above Example 3 is the best result obtained after many trials and experiments. During the experiment, sodium citrate boiling water heating repair method, sodium citrate microwave heating repair method, EDTA microwave heating repair method, and EDTA high pressure heating repair method were also used to perform the above antigen repair operation.

[0047] Refer to the following Comparative Examples 4 to 9; Comparative Example 4 The method is substantially the same as Example 3, except that 200 mL of 1x sodium citrate antigen retrieval solution is used for retrieval in a boiling water bath for 15 min.

[0048] The results refer to Fig. 7A .

[0049] Comparative Example 5 The method is substantially the same as Example 3, except that 200 mL of 1x sodium citrate antigen retrieval solution is used for retrieval at low temperature in a microwave oven for 10 min.

[0050] The low-fire parameters are microwave power: 300w, microwave frequency: 2450 MHz; The results refer to Figure 7B .

[0051] Comparative Example 6 The method is substantially the same as Example 3, except that 200 mL of 1x sodium citrate antigen retrieval solution is used for retrieval at low temperature in a microwave oven for 15 min.

[0052] The low-fire parameters are microwave power: 300w, microwave frequency: 2450 MHz; The results refer to Figure 7C .

[0053] pass FIG. 7A to FIG. 7C It can be seen that the morphology of positive muscle fibers was unclear after immunofluorescence staining after antigen retrieval with sodium citrate.

[0054] Comparative Example 7 The method is generally the same as Example 3, except that EDTA antigen repair solution is used for repair in a boiling water bath for 15 minutes.

[0055] The results refer to Fig. 8A , repaired with EDTA antigen repair solution in a boiling water bath for 15 min, the morphology of positive muscle fibers was not clear.

[0056] Comparative Example 8 The method is generally the same as Example 3, except that EDTA antigen repair solution is used for repair at low temperature in a microwave oven for 15 minutes.

[0057] The results refer to Figure 8BAfter repairing at low heat in a microwave oven for 15 minutes, since the EDTA antigen repair solution is an alkaline solution, too long a repair time will cause changes in the morphology of the muscle fibers.

[0058] Comparative Example 9 In the antigen retrieval step, since high-pressure retrieval is sensitive to tissue sections, the high-pressure retrieval time was shortened during the exploration of high-pressure retrieval conditions for samples using a pressure cooker. High-pressure retrieval was performed using EDTA antigen retrieval solution for 3 minutes (Figure 9 A) and high-pressure retrieval was performed using sodium citrate antigen retrieval solution for 10 minutes (Figure 9 B). Fig. 9A and Fig. 9B The results showed that after high-pressure repair, the morphology of positive muscle fibers was unclear, and after 10 minutes of high-pressure repair, the morphology of muscle fibers changed.

[0059] It can be seen from the above-mentioned Example 3 and Comparative Examples 4 to 9 that the positive muscle fiber morphology is brighter and clearer after repairing with EDTA antigen repair solution at low temperature in a microwave oven for 10 minutes.

[0060] Part III: Verification of the effect of different antigen retrieval methods on the staining of fresh muscle tissue samples (1) Sample fixation In this experiment, the quadriceps femoris muscle of Lufeng cattle was selected, and the slaughter time was within 30 minutes; The fixing method is: Cut along the direction of muscle fibers into 1*1*0.5 cm cross sections according to the length*width*height specifications 3 For muscle samples of different sizes, immerse the muscles in 10wt% neutral formalin (the volume of 10% neutral formalin should not be less than 20 times the volume of the tissue sample) and fix them at 4℃ for 16 h.

[0061] (2) Slicing After fixation, the sample was transferred to a centrifuge tube filled with 70 vol% alcohol and washed three times, 5 min / time. The sample dehydration, OTC embedding and sectioning steps were referred to Example 1.

[0062] (3) Dyeing Dyeing reference example 3.

[0063] FIG. 10A to FIG. 10C The antigen retrieval method represented can be referred to Example 3; Fig. 10D The antigen retrieval methods represented are: staining photos repaired in a boiling water bath for 10 min using EDTA antigen retrieval solution; Fig. 10E The antigen retrieval methods represented are: stained photos repaired in a boiling water bath for 10 min using sodium citrate antigen retrieval solution; Fig.10F The antigen retrieval method represented is: staining photos of using sodium citrate antigen retrieval solution to retrieval at low heat in a microwave oven for 10 minutes.

[0064] This section proves that the commonly used repair methods have relatively good repair effects on fresh samples.

[0065] The innovation of the present invention lies in that, through repeated exploration, the fixation process and the staining process are optimized, so that even after the samples are preserved for up to 180 days, good fixation and staining effects can still be obtained. This research effectively solves the problem of the need to process samples immediately in the existing process, is conducive to the long-term storage and transportation of samples, and reduces the storage requirements for sample testing.

[0066] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for fixing muscle tissue suitable for transportation, characterized in that: The freshly obtained muscle samples were fixed in a formalin solution with a concentration of 8-12wt% for 14-18h; then, the fixed muscle samples were immersed in an ethanol solution with a concentration of 60-80vol% for preservation.

2. The method for fixing muscle tissue for transportation according to claim 1, characterized in that: The freshly obtained muscle samples were fixed in a 10wt% formalin solution for 16 hours; then, the fixed muscle samples were immersed in a 70vol% ethanol solution for preservation.

3. The method for fixing muscle tissue for transportation according to claim 1 or 2, characterized in that: The storage temperature is 4℃.

4. The method for fixing muscle tissue for transportation according to claim 1 or 2, characterized in that: The dimensions of the muscle sample are: 0.8-1.2 cm in length, 0.8-1.2 cm in width, and 0.3-1.0 cm in height.

5. A method for fluorescent staining of muscle tissue, characterized in that: The muscle sample is first fixed by the method described in any one of claims 1 to 4; and then the fixed muscle sample is sent to a laboratory for sectioning and fluorescent staining.

6. The method according to claim 5, characterized in that The slicing method is: The muscle samples were removed from the ethanol solution and dehydrated; The dehydrated muscle samples were then embedded and frozen using OTC embedding medium and sectioned.

7. The method according to claim 5, characterized in that The method of the fluorescent staining is: The tissue sections after sectioning are fixed, antigen repaired, blocked, first incubated with primary antibody, first incubated with secondary antibody, first incubated with dye, blocked, second incubated with primary antibody, second incubated with secondary antibody, second incubated with dye, and then sealed and tested.

8. The method according to claim 7, characterized in that The antigen repair agent used in the antigen repair is EDTA. The specific operation of antigen repair is: placing the fixed tissue section in EDTA antigen repair solution, heating it in a microwave heating mode at low heat for 10 minutes, and naturally cooling it to room temperature.

9. The method according to claim 7, characterized in that: The primary antibody used in the first incubation was BF-35 primary antibody; the secondary antibody used in the first incubation was HRP secondary antibody; the primary antibody used in the second incubation was SC-71 primary antibody; the secondary antibody used in the second incubation was HRP secondary antibody; The dye used in the first incubation was TSA-if488 staining working solution; the dye used in the second incubation was TSA-CY3 staining working solution.

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