CN116990101A - Pretreatment method for easily-fallen tissues and multiple immunofluorescence staining method thereof - Google Patents
Pretreatment method for easily-fallen tissues and multiple immunofluorescence staining method thereof Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000003125 immunofluorescent labeling Methods 0.000 title claims abstract description 27
- 238000002203 pretreatment Methods 0.000 title claims abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 79
- 210000001519 tissue Anatomy 0.000 claims description 77
- 235000019441 ethanol Nutrition 0.000 claims description 30
- 239000003795 chemical substances by application Substances 0.000 claims description 29
- 238000002791 soaking Methods 0.000 claims description 29
- 238000011534 incubation Methods 0.000 claims description 22
- 238000010186 staining Methods 0.000 claims description 22
- 239000000427 antigen Substances 0.000 claims description 20
- 102000036639 antigens Human genes 0.000 claims description 20
- 108091007433 antigens Proteins 0.000 claims description 20
- 210000002435 tendon Anatomy 0.000 claims description 13
- 239000003480 eluent Substances 0.000 claims description 11
- 210000000988 bone and bone Anatomy 0.000 claims description 10
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 9
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- 210000000845 cartilage Anatomy 0.000 claims description 9
- 238000007789 sealing Methods 0.000 claims description 5
- 239000003431 cross linking reagent Substances 0.000 claims description 4
- 239000000341 volatile oil Substances 0.000 claims description 4
- 150000008280 chlorinated hydrocarbons Chemical class 0.000 claims description 3
- 230000001678 irradiating effect Effects 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000004132 cross linking Methods 0.000 abstract description 75
- 230000007170 pathology Effects 0.000 abstract description 4
- 239000012188 paraffin wax Substances 0.000 description 8
- 230000036571 hydration Effects 0.000 description 7
- 238000006703 hydration reaction Methods 0.000 description 7
- 238000004043 dyeing Methods 0.000 description 6
- 239000011022 opal Substances 0.000 description 5
- 230000000903 blocking effect Effects 0.000 description 4
- 230000000887 hydrating effect Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 230000003321 amplification Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 238000000116 DAPI staining Methods 0.000 description 1
- 206010069364 Meniscal degeneration Diseases 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
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- 230000008023 solidification Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
-
- 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
Abstract
The invention provides a pretreatment method of easily-dropped tissues and a multiple immunofluorescence staining method thereof, belonging to the technical field of biomedical experimental pathology. The pretreatment method of the tissue easy to fall off adopts the steps of pulling out the tissue and then directly carrying out photosensitive crosslinking under ultraviolet irradiation, combines the specific irradiation time to ensure that the obtained slice is firm, avoids the step of baking the slice, and simultaneously solves the problem that the tissue easy to fall off in multiple immunofluorescence staining is easy to fall off. The multiple immunofluorescence staining method can shorten the program time and improve the treatment efficiency.
Description
Technical Field
The invention belongs to the technical field of biomedical experimental pathology, and particularly relates to a pretreatment method of easily-fallen tissues and a multiple immunofluorescence staining method thereof.
Background
In the current experimental pathology technology, paraffin section immunological staining can be subjected to an antigen retrieval process, and the process usually leads to the condition that tissue sections such as cartilage, bone, tendon and the like are easy to fall off, so that the tissue structure integrity is seriously affected.
In recent years, the emerging multiple immunofluorescent staining technique is widely used because it is not limited by the species of the antibody, and a common label of 6 to 8 antibodies can be performed on one paraffin section. This technique is mainly derived from the tyrosine amplification technique (Tyramide signal amplification, TSA) published by Zsuzsanana Tth and É va Mezey, and the technical scheme is shown in FIG. 1. In multiplex immunofluorescent staining, antigen retrieval and microwaving steps to remove antibodies are necessary. Along with the increase of detection targets, tissue sections need to be subjected to microwave treatment for one round, and under the treatment mode, the tissue integrity of the tissue is difficult to be ensured due to the fact that cartilage, bones, tendons and the like are easy to fall off, so that multiple immunofluorescence staining of the tissue which is easy to fall off is difficult to develop at present. The method now supports a cartilage tissue research article (Cellular features of localized microenvironments in human meniscal degeneration: a single-cell transcriptomic study, eLife,2022 Dec 22;11:e79585.doi: 10.7554/eLife.79585) which reports the first cartilage tissue examined for mIHC staining. Bone tissue and tendon tissue have also been subjected to mhic experiments, and have not been reported in the related art literature.
In conclusion, the multiple immunofluorescent staining (mhic) technology is widely applied to the technical field of experimental pathology, but the technology can undergo an antigen retrieval process and also can carry out microwave treatment for multiple times to remove antibodies, so that the tissue which is easy to fall off is difficult to apply in the mhic. Therefore, how to apply the easily-dropped piece tissue to multiple immunofluorescence staining is a problem to be solved urgently.
Disclosure of Invention
The invention mainly aims to provide a pretreatment method of easily-fallen tissues and a multiple immunofluorescence staining method thereof, so as to overcome the defects in the prior art.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention comprises the following steps:
according to a first aspect of an embodiment of the present invention, there is provided a pretreatment method of an easy-to-fall tissue, including the steps of:
fishing out the tissue slice, collecting the slice, and irradiating the slice with ultraviolet for 1-5 min to obtain a cured slice;
dewaxing the solidified slice to finish pretreatment of the tissue easy to fall off;
the water agent adopted by the fishing slice contains oily photosensitive cross-linking agent.
Preferably, the tissue section is derived from cartilage, bone or tendon.
Preferably, both the front and back sides of the slice are irradiated during the ultraviolet irradiation.
Preferably, the dewaxing comprises the steps of:
and soaking the solidified slice in the dewaxing agent, absolute ethyl alcohol, 95-99.99% v/v ethyl alcohol, 90-95% v/v ethyl alcohol and 70-90% v/v ethyl alcohol in sequence.
Preferably, the dewaxing agent is one or more selected from benzene dewaxing agent, toluene dewaxing agent, chlorinated hydrocarbon dewaxing agent and essential oil dewaxing agent.
Preferably, the soaking times of the dewaxing agent are 2-4 times;
the soaking times of the absolute ethyl alcohol, the 95-99.99% v/v ethyl alcohol, the 90-95% v/v ethyl alcohol and the 70-90% v/v ethyl alcohol are 1-3 times.
The invention also provides application of the pretreatment method of the easily-dropped tissue in tissue multiplex immunofluorescence staining.
The invention also provides a multiple immunofluorescence staining method of the tissue easy to fall, which comprises the following steps:
the method is adopted to pretreat the tissue easy to fall off to obtain a sample to be dyed;
and (3) sequentially carrying out antigen repair, sealing, primary antibody incubation, secondary antibody incubation, fluorescent group incubation, antibody removal and staining on the sample to be stained, and finishing multiple immunofluorescence staining of the tissue easy to fall.
Preferably, the antigen retrieval specifically comprises the following steps:
and mixing the sample to be dyed with the antibody eluent, and repairing for 2-4 min.
Preferably, the repair uses high fire conditions;
the power of the high fire condition is 600-700W.
Compared with the prior art, the invention has the advantages that:
the invention provides a pretreatment method of easily-dropped piece tissues and a multiple immunofluorescence staining method thereof, wherein the pretreatment method adopts the steps of fishing pieces and then directly carrying out photosensitive crosslinking under ultraviolet irradiation, and the obtained slices are firm by combining with specific irradiation time, so that the problem that easily-dropped piece tissues are easy to drop in multiple immunofluorescence staining is solved.
In addition, the multiple immunofluorescence staining method of the invention greatly shortens the program time and improves the processing efficiency in the antigen repairing process.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic flow diagram of a prior art tyrosine amplification technique published by Zsuzsanana Tth and É va Mezey;
fig. 2 is a graph showing the results of staining of the experimental group for different sheet baking times after dewaxing and rehydration, wherein: FIG. 2 (a) shows the result of directly staining the baked sheet for 0h, i.e., the unbaked sheet; FIG. 2 (b) shows the result of baking sheets for 4-10 hours; FIG. 2 (c) shows the results of baking sheets for 18-24 hours;
fig. 3 is a graph showing the results of dyeing of the experimental groups for different photocrosslinking times after dewaxing and rehydration, wherein: FIG. 3 (a) shows the result of irradiation for 0s, i.e., non-irradiation, of the photosensitive crosslinking time; FIG. 3 (b) is a result of irradiation for 20s at a photosensitive crosslinking time; FIG. 3 (c) is a result of irradiation for 40s at a photosensitive crosslinking time; FIG. 3 (d) is a result of irradiation for 1min for a photo-sensitive crosslinking time; FIG. 3 (e) is the result of irradiation for 2min for the photosensitive crosslinking time; FIG. 3 (f) is a result of irradiation for 3min for a photo-sensitive crosslinking time; FIG. 3 (g) is a result of irradiation for 4min for a photo-sensitive crosslinking time; FIG. 3 (h) is a result of irradiation for 5min for a photo-sensitive crosslinking time;
fig. 4 is a graph showing the results of staining of experimental groups with different sheet baking times after the first microwave treatment, wherein: FIG. 4 (a) shows the result of baking sheets 0h, i.e., not baking sheets; FIG. 4 (b) shows the result of baking sheets for 4-10 hours; FIG. 4 (c) shows the results of baking sheets for 18-24 hours;
fig. 5 is a graph of experimental group staining results for different photosensitive crosslinking times after the first microwave treatment, wherein: FIG. 5 (a) is a result of irradiation for 20s at a photosensitive crosslinking time; FIG. 5 (b) is a result of irradiation for 40s at a photosensitive crosslinking time; FIG. 5 (c) is a result of irradiation for 1min for a photo-sensitive crosslinking time; FIG. 5 (d) is the result of irradiation for 2min for the photosensitive crosslinking time; FIG. 5 (e) is the result of irradiation for 3min with photosensitive crosslinking time; FIG. 5 (f) is the result of irradiation for 4min with photosensitive crosslinking time; FIG. 5 (g) is a result of irradiation for 5min for a photo-sensitive crosslinking time;
fig. 6 is a graph showing the staining results of the experimental groups for different sheet baking times after the 2 nd microwave treatment, wherein: FIG. 6 (a) shows the result of baking sheets 0h, i.e., not baking sheets; FIG. 6 (b) shows the result of baking sheets for 4-10 hours; FIG. 6 (c) shows the results of baking sheets for 18-24 hours;
fig. 7 is a graph showing the results of staining of experimental groups with different photosensitive crosslinking times after the 2 nd microwave treatment, wherein: FIG. 7 (a) is a result of irradiation for 20s at a photosensitive crosslinking time; FIG. 7 (b) is a result of irradiation for 40s at a photosensitive crosslinking time; FIG. 7 (c) is a result of irradiation for 1min for a photo-sensitive crosslinking time; FIG. 7 (d) is a result of irradiation for 2min for a photo-sensitive crosslinking time; FIG. 7 (e) is a result of irradiation for 3min for a photo-sensitive crosslinking time; FIG. 7 (f) is a result of irradiation for 4min for a photo-sensitive crosslinking time; FIG. 7 (g) is a result of irradiation for 5min for a photo-sensitive crosslinking time;
fig. 8 shows the results of the staining of the experimental groups for different sheet baking times after the 7 th microwave treatment, wherein: FIG. 8 (a) shows the result of baking sheets 0h, i.e., not baking sheets; FIG. 8 (b) shows the result of baking sheets for 4-10 hours; FIG. 8 (c) shows the results of baking sheets for 18-24 hours;
fig. 9 shows the results of the experimental group staining for different photosensitive crosslinking times after the 7 th microwave treatment, wherein: FIG. 9 (a) is a result of irradiation for 20s at a photosensitive crosslinking time; FIG. 9 (b) is a result of irradiation for 40s at a photosensitive crosslinking time; FIG. 9 (c) is a result of irradiation for 1min for a photo-sensitive crosslinking time; FIG. 9 (d) is a result of irradiation for 2min for a photo-sensitive crosslinking time; FIG. 9 (e) is a result of irradiation for 3min for a photo-sensitive crosslinking time; FIG. 9 (f) is a result of irradiation for 4min for a photo-sensitive crosslinking time; FIG. 9 (g) is a result of irradiation for 5min for a photo-sensitive crosslinking time;
fig. 10 is a bone Opal 7 color fluorescence picture (1);
FIG. 11 is a bone Opal 7 color fluorescence image (2);
FIG. 12 is a fluorescent photograph of tendon Opal 4 color;
fig. 13 is a fluorescent photograph of tendon Opal 7 color.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
In a first aspect of the embodiment of the present invention, a pretreatment method for an easy-to-fall tissue is provided, including the following steps:
fishing out the tissue slice, collecting the slice, and irradiating the slice with ultraviolet for 1-5 min to obtain a cured slice;
dewaxing the solidified slice to finish pretreatment of the tissue easy to fall off;
the water agent adopted by the slice fishing contains the oily photosensitive cross-linking agent, and the oily photosensitive cross-linking agent is automatically coated on the surface of the slice in the slice fishing process, meanwhile, the slice baking step is avoided, the ultraviolet irradiation is directly carried out for a specific time period, the solidification of the slice is realized, and the stability of the tissue easy to fall off is improved; the reagent used for the scooping sheet is preferably an Adhesive-Coated Slide available from Leica company.
In the present invention, the tissue slice is preferably derived from cartilage, bone or tendon, and both the front and back sides of the slice are preferably irradiated during the ultraviolet irradiation, and the irradiation time per side is preferably 1 to 2 minutes.
The dewaxing according to the present invention preferably comprises the steps of: soaking the solidified slice sequentially by adopting a dewaxing agent, absolute ethyl alcohol, 95-99.99% v/v ethyl alcohol, 90-95% v/v ethyl alcohol and 70-90% v/v ethyl alcohol, wherein the dewaxing agent is preferably one or more of benzene dewaxing agent, toluene dewaxing agent, chlorinated hydrocarbon dewaxing agent or essential oil dewaxing agent, more preferably essential oil dewaxing agent, and the soaking frequency of the dewaxing agent is preferably 2-4 times; the soaking times of the absolute ethyl alcohol, the 95-99.99% v/v ethyl alcohol, the 90-95% v/v ethyl alcohol and the 70-90% v/v ethyl alcohol are preferably 1-3 times.
The invention also provides application of the pretreatment method of the tissue easy to fall in multiple immunofluorescence staining of the tissue, and the pretreatment method can avoid the situation that the tissue integrity of the tissue easy to fall is difficult to ensure due to the fact that antibodies are removed through multiple antigen repair and microwave treatment in multiple immunofluorescence staining.
The invention also provides a multiple immunofluorescence staining method of the tissue easy to fall, which comprises the following steps: the method is adopted to pretreat the tissue easy to fall off to obtain a sample to be dyed; sequentially carrying out antigen repair, sealing, primary anti-incubation, secondary anti-incubation, fluorescent group incubation, removing antibodies by microwave treatment and staining on a sample to be stained to finish multiple immunofluorescence staining of the tissue easy to fall, wherein the antigen repair preferably specifically comprises the following steps: mixing a sample to be dyed with an antibody eluent, and repairing for 2-4 min, wherein the antibody eluent is preferably AbCracker, LOT X1KCJ10A, and the repairing is preferably performed under a high fire condition; the power of the high fire condition is 600-700W.
The present invention is discussed in detail below in conjunction with specific embodiments to facilitate understanding of the technical solutions of the present invention.
Example 1
(1) Selecting tendon tissues to be detected, and slicing the tissues to be detected, wherein the thickness of the tissue slices to be detected is 3-5 mu m for later use;
(2) After slicing is completed in the step (1), fishing out slices by using an Adhesive-Coated Slide purchased from Leica company, and then carrying out photosensitive crosslinking for 1min under ultraviolet irradiation;
(3) Dewaxing and hydrating: sequentially soaking the sample in TO biological transparent agent twice, soaking the sample in absolute ethyl alcohol twice, soaking the sample in 95% v/v ethanol once, soaking the sample in 90% v/v ethanol once and soaking the sample in 80% v/v ethanol once TO obtain dewaxed and hydrated tissue as a sample TO be dyed;
(4) And sequentially carrying out antigen repair, blocking, primary antibody incubation, secondary antibody incubation, fluorescent group incubation, antibody removal by microwave treatment and staining on the sample to be stained.
Wherein, antigen retrieval is carried out by adopting antibody eluent (AbCracker, LOT X1KCJ 10A) for 3min of high fire (700W).
Example 2
(1) Selecting bone tissue to be detected, and slicing the tissue to be detected, wherein the thickness of the tissue slice to be detected is 3-5 mu m for later use;
(2) After slicing is completed in the step (1), fishing out slices by using an Adhesive-Coated Slide purchased from Leica company, and then carrying out photosensitive crosslinking for 2min under ultraviolet irradiation;
(3) Dewaxing and hydrating: sequentially soaking the sample in TO biological transparent agent twice, soaking the sample in absolute ethyl alcohol twice, soaking the sample in 95% v/v ethanol once, soaking the sample in 90% v/v ethanol once and soaking the sample in 80% v/v ethanol once TO obtain dewaxed and hydrated tissue as a sample TO be dyed;
(4) And sequentially carrying out antigen repair, blocking, primary antibody incubation, secondary antibody incubation, fluorescent group incubation, antibody removal by microwave treatment and staining on the sample to be stained, namely multiplex fluorescent staining.
Wherein, antigen retrieval is carried out by adopting antibody eluent (AbCracker, LOT X1KCJ 10A) for 3min of high fire (700W).
The final staining results are shown in fig. 10 and 11, which show that the tissue is intact and the sheets are not dropped.
Example 3
(1) Selecting cartilage tissue to be detected, and slicing the tissue to be detected, wherein the thickness of the tissue slice to be detected is 3-5 mu m for later use;
(2) After slicing is completed in the step (1), fishing out slices by using an Adhesive-Coated Slide purchased from Leica company, and then carrying out photosensitive crosslinking for 3min under ultraviolet irradiation;
(3) Dewaxing and hydrating: sequentially soaking the sample in TO biological transparent agent twice, soaking the sample in absolute ethyl alcohol twice, soaking the sample in 95% v/v ethanol once, soaking the sample in 90% v/v ethanol once and soaking the sample in 80% v/v ethanol once TO obtain dewaxed and hydrated tissue as a sample TO be dyed;
(4) And sequentially carrying out antigen repair, blocking, primary antibody incubation, secondary antibody incubation, fluorescent group incubation, antibody removal by microwave treatment and staining on the sample to be stained.
Wherein, antigen retrieval is carried out by adopting antibody eluent (AbCracker, LOT X1KCJ 10A) for 3min of high fire (700W).
Example 4
(1) Selecting tendon tissues to be detected, and slicing the tissues to be detected, wherein the thickness of the tissue slices to be detected is 3-5 mu m for later use;
(2) After slicing is completed in the step (1), fishing out slices by using an Adhesive-Coated Slide purchased from Leica company, and then carrying out photosensitive crosslinking for 4min under ultraviolet irradiation;
(3) Dewaxing and hydrating: soaking twice sequentially by using TO biological transparent agent, soaking twice by using absolute ethyl alcohol, soaking once by using 95% v/v ethyl alcohol, soaking once by using 90% v/v ethyl alcohol, and soaking once by using 80% v/v ethyl alcohol TO obtain dewaxed and hydrated tendon tissues as samples TO be dyed;
(4) And sequentially carrying out antigen repair, blocking, primary antibody incubation, secondary antibody incubation, fluorescent group incubation, antibody removal by microwave treatment and staining on the sample to be stained, namely multiplex fluorescent staining.
Wherein, antigen retrieval is carried out by adopting antibody eluent (AbCracker, LOT X1KCJ 10A) for 3min of high fire (700W).
The final staining results are shown in fig. 12 and 13, which show that the tissue is intact and the sheets are not dropped.
Experimental example 1
1. Based on the procedure of example 1, the other conditions were kept unchanged, and individual conditions therein were replaced, designed as the following control experiments, the methods of operation were as follows:
after paraffin sections of control (1), dewaxing hydration is directly carried out, and dyeing is carried out (dewaxing hydration step is the same as in example 1);
after paraffin sections of control (2), the sections were baked for 4 to 10 hours and subjected to dewaxing and hydration to be dyed (dewaxing and hydration steps were the same as in example 1).
After paraffin sections of control (3), the baking time was prolonged to 18-24 hours, and dewaxing hydration was performed to dye (dewaxing hydration step was the same as in example 1).
The photosensitive crosslinking time is replaced by 0, 20s, 40s, 1min, 2min, 3min, 4min and 5min respectively in the control (4).
In order to intuitively show the integrity of the tissue in the immunofluorescence multi-staining experiment, the experimental conditions of microwave treatment which are most likely to cause flaking in the experimental step are simulated, wherein the microwave treatment is carried out for 16 minutes for the first time, and the microwave heating is stopped after each treatment until the microwave heating is boiling.
The dewaxed and hydrated samples in each group were processed as follows:
(1) antigen retrieval: microwave high fire 700W for 16min (example 1 replaced with treatment with antibody eluent (AbCracker, LOT X1KCJ 10A), high fire 700W for 3 min);
(2) cooling to room temperature, washing off repair liquid, sealing, and RT for 10min;
(3) incubation with primary antibody, PBS wash 5 times, 2 min/time;
(4) incubating with a secondary antibody, and carrying out RT for 10min;
(5) washing with PBS for 5 times and 2 min/time;
(6) incubating with Opal fluorescein, and performing RT for 10min;
(7) washing with PBS for 5 times and 2 min/time;
(8) adding antibody eluent, and microwave heating to boil;
(9) repeating the steps (2) - (8) until the 7 th microwave is boiled, cooling to room temperature, and washing off eluent;
DAPI staining and sealing.
The invention respectively carries out HE staining on tissues after dewaxing and rehydration and each microwave treatment, and respectively records the following four stages through panorama acquisition: (1) dewaxing and rehydrating, (2) first microwave treatment, (3) 2 nd microwave treatment and (4) 7 th microwave treatment.
HE staining results
(1) The results of the staining of the experimental groups after various baking times after dewaxing and rehydration of each group are shown in fig. 2, wherein:
FIG. 2 (a) shows the result of directly staining the baked sheet for 0h, i.e., the unbaked sheet;
FIG. 2 (b) shows the result of baking sheets for 4-10 hours;
FIG. 2 (c) shows the results of baking sheets for 18-24 hours;
(2) After dewaxing and rehydration of each group, the groups underwent different photosensitive crosslinking times, and the dyeing results are shown in fig. 3, wherein:
FIG. 3 (a) shows the result of irradiation for 0s, i.e., non-irradiation, of the photosensitive crosslinking time;
FIG. 3 (b) is a result of irradiation for 20s at a photosensitive crosslinking time;
FIG. 3 (c) is a result of irradiation for 40s at a photosensitive crosslinking time;
FIG. 3 (d) is a result of irradiation for 1min for a photo-sensitive crosslinking time;
FIG. 3 (e) is the result of irradiation for 2min for the photosensitive crosslinking time;
FIG. 3 (f) is a result of irradiation for 3min for a photo-sensitive crosslinking time;
FIG. 3 (g) is a result of irradiation for 4min for a photo-sensitive crosslinking time;
FIG. 3 (h) is a result of irradiation for 5min for a photo-sensitive crosslinking time;
(3) The experimental groups, which underwent different baking times after the first microwave treatment, were stained as shown in fig. 4, in which:
FIG. 4 (a) shows the result of baking sheets 0h, i.e., not baking sheets;
FIG. 4 (b) shows the result of baking sheets for 4-10 hours;
FIG. 4 (c) shows the results of baking sheets for 18-24 hours;
(4) After the first microwave treatment, each group was subjected to an experimental group of different photosensitive crosslinking times, and the dyeing results are shown in fig. 5, in which:
FIG. 5 (a) is a result of irradiation for 20s at a photosensitive crosslinking time;
FIG. 5 (b) is a result of irradiation for 40s at a photosensitive crosslinking time;
FIG. 5 (c) is a result of irradiation for 1min for a photo-sensitive crosslinking time;
FIG. 5 (d) is the result of irradiation for 2min for the photosensitive crosslinking time;
FIG. 5 (e) is the result of irradiation for 3min with photosensitive crosslinking time;
FIG. 5 (f) is the result of irradiation for 4min with photosensitive crosslinking time;
FIG. 5 (g) is a result of irradiation for 5min for a photo-sensitive crosslinking time;
(5) The experimental groups, which underwent different baking times after the 2 nd microwave treatment of each group, were stained as shown in fig. 6, wherein:
FIG. 6 (a) shows the result of baking sheets 0h, i.e., not baking sheets;
FIG. 6 (b) shows the result of baking sheets for 4-10 hours;
FIG. 6 (c) shows the results of baking sheets for 18-24 hours;
(6) After the 2 nd microwave treatment of each group, the experimental groups with different photosensitive crosslinking time were subjected to the dyeing results shown in fig. 7, wherein:
FIG. 7 (a) is a result of irradiation for 20s at a photosensitive crosslinking time;
FIG. 7 (b) is a result of irradiation for 40s at a photosensitive crosslinking time;
FIG. 7 (c) is a result of irradiation for 1min for a photo-sensitive crosslinking time;
FIG. 7 (d) is a result of irradiation for 2min for a photo-sensitive crosslinking time;
FIG. 7 (e) is a result of irradiation for 3min for a photo-sensitive crosslinking time;
FIG. 7 (f) is a result of irradiation for 4min for a photo-sensitive crosslinking time;
FIG. 7 (g) is a result of irradiation for 5min for a photo-sensitive crosslinking time;
(7) The experimental groups, which underwent different baking times after the 7 th microwave treatment of each group, were stained as shown in fig. 8, wherein:
FIG. 8 (a) shows the result of baking sheets 0h, i.e., not baking sheets;
FIG. 8 (b) shows the result of baking sheets for 4-10 hours;
FIG. 8 (c) shows the results of baking sheets for 18-24 hours;
(8) The experimental groups, which underwent different photo-sensitive crosslinking times after the 7 th microwave treatment of each group, were stained as shown in fig. 9, wherein:
FIG. 9 (a) is a result of irradiation for 20s at a photosensitive crosslinking time;
FIG. 9 (b) is a result of irradiation for 40s at a photosensitive crosslinking time;
FIG. 9 (c) is a result of irradiation for 1min for a photo-sensitive crosslinking time;
FIG. 9 (d) is a result of irradiation for 2min for a photo-sensitive crosslinking time;
FIG. 9 (e) is a result of irradiation for 3min for a photo-sensitive crosslinking time;
FIG. 9 (f) is a result of irradiation for 4min for a photo-sensitive crosslinking time;
FIG. 9 (g) is a result of irradiation for 5min for a photo-sensitive crosslinking time;
3. interpretation of the dyeing results
The samples adopted by the invention are continuous paraffin sections of tendon tissues, so that the initial state is very close, and the contrast effect of different experimental conditions can be better represented.
As can be seen from fig. 2, 4, 6 and 8, the conventional use in the prior art: the method of dewaxing hydration directly after paraffin slicing and roasting slices for 4-10 hours after paraffin slicing can not avoid slice falling phenomenon of easily falling tissues, can not keep complete tissue structures, and is more serious with the increase of microwave treatment times; meanwhile, as shown in fig. 3, 5, 7 and 9, the result of the scheme provided by the invention (the graph c-f in each graph) adopting the ultraviolet crosslinking time of 1-4 min shows that the scheme provided by the invention can keep a complete tissue structure and cannot fall off along with the increase of microwave treatment times.
And it is also known that: the prior art generally increases the slice baking time to prevent tissue slice falling, but the method is not suitable for tendon, bone, cartilage and other tissues easy to slice falling, and can be shown in four groups of pictures in fig. 2, 4, 6 and 8: the increase in sheet baking time does not reduce tissue flaking.
As can be seen from fig. 3, fig. 5, fig. 7 and fig. 9, the optimal time of ultraviolet crosslinking is 1-4 min, the tissue integrity cannot be ensured even if the time is too long or too short, the optimal ultraviolet crosslinking time is matched with the treatment steps of the invention, the optimal anti-falling result can be obtained, and fig. 9 shows that the treatment method provided by the invention can still keep the tissue integrity after seventh microwave.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. The pretreatment method of the easy-falling tissue is characterized by comprising the following steps of:
fishing out the tissue slice, collecting the slice, and irradiating the slice with ultraviolet for 1-5 min to obtain a cured slice;
dewaxing the solidified slice to finish pretreatment of the tissue easy to fall off;
the water agent adopted by the fishing slice contains oily photosensitive cross-linking agent.
2. The method of claim 1, wherein the tissue section is derived from cartilage, bone or tendon.
3. The method for pretreating a tissue susceptible to chipping according to claim 1 wherein both sides of the slice are irradiated during said ultraviolet irradiation.
4. The method of pretreatment of fugitive sheet tissue according to claim 1, wherein said dewaxing comprises the steps of:
and soaking the solidified slice in the dewaxing agent, absolute ethyl alcohol, 95-99.99% v/v ethyl alcohol, 90-95% v/v ethyl alcohol and 70-90% v/v ethyl alcohol in sequence.
5. The pretreatment method of the easily removable piece tissue according to claim 4, wherein the dewaxing agent is one or more selected from benzene dewaxing agent, toluene dewaxing agent, chlorinated hydrocarbon dewaxing agent and essential oil dewaxing agent.
6. The pretreatment method of easy-to-fall tissue according to claim 4, wherein the soaking times of the dewaxing agent are 2-4 times;
the soaking times of the absolute ethyl alcohol, the 95-99.99% v/v ethyl alcohol, the 90-95% v/v ethyl alcohol and the 70-90% v/v ethyl alcohol are 1-3 times.
7. The use of the pretreatment method of the easily fallen tissue according to any one of claims 1-6 in multiplex immunofluorescence staining of tissue.
8. The multiple immunofluorescence staining method for the easily-dropped tissue is characterized by comprising the following steps of:
pretreating the easily fallen tissues by adopting the method of any one of claims 1-6 to obtain a sample to be dyed;
and (3) sequentially carrying out antigen repair, sealing, primary antibody incubation, secondary antibody incubation, fluorescent group incubation, antibody removal and staining on the sample to be stained, and finishing multiple immunofluorescence staining of the tissue easy to fall.
9. The multiple immunofluorescent staining method of easily removable tissues according to claim 8, wherein the antigen retrieval specifically comprises the steps of:
and mixing the sample to be dyed with the antibody eluent, and repairing for 2-4 min.
10. The multiple immunofluorescent staining method of easily removable tissue according to claim 8, wherein the repairing is performed under high fire conditions;
the power of the high fire condition is 600-700W.
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