CN112067409A - Improved H.E staining method for mouse bone marrow tissue section - Google Patents
Improved H.E staining method for mouse bone marrow tissue section Download PDFInfo
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- 210000001519 tissue Anatomy 0.000 title claims abstract description 50
- 210000001185 bone marrow Anatomy 0.000 title claims abstract description 25
- 238000007447 staining method Methods 0.000 title claims abstract description 18
- 238000010186 staining Methods 0.000 claims abstract description 35
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 11
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims abstract description 6
- 235000017557 sodium bicarbonate Nutrition 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 14
- 230000000694 effects Effects 0.000 abstract description 10
- 238000004043 dyeing Methods 0.000 abstract description 9
- 238000002791 soaking Methods 0.000 abstract description 9
- 239000012188 paraffin wax Substances 0.000 abstract description 6
- 239000000243 solution Substances 0.000 abstract description 6
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 abstract description 5
- 239000012192 staining solution Substances 0.000 abstract description 5
- 238000005406 washing Methods 0.000 abstract description 4
- 210000000988 bone and bone Anatomy 0.000 abstract description 3
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 abstract description 2
- 239000012153 distilled water Substances 0.000 abstract description 2
- 239000008096 xylene Substances 0.000 abstract description 2
- 230000007935 neutral effect Effects 0.000 description 16
- 239000011521 glass Substances 0.000 description 12
- 239000006059 cover glass Substances 0.000 description 8
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 6
- 230000006872 improvement Effects 0.000 description 5
- 238000007598 dipping method Methods 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 2
- 210000002798 bone marrow cell Anatomy 0.000 description 2
- 210000001669 bursa of fabricius Anatomy 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108010077544 Chromatin Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 230000031877 prophase Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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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
-
- 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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- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
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Abstract
The invention discloses an improved H.E staining method for mouse marrow tissue slices, which is characterized in that paraffin is fully dissolved by properly increasing the time length of a previous xylene dewaxing transparent step, so that staining solution fully enters tissues during staining; meanwhile, the concentration gradient of the alcohol and the time for soaking and dehydrating the high-concentration alcohol are reduced, and the softened bone tissue can be effectively prevented from shrinking and hardening to influence the slicing quality; the slice is blued by using 2% sodium bicarbonate in alkalescent solution, so that the blueing efficiency and effect are improved, and the cost is reduced; and the dyeing effect of the eosin solution can be improved by washing with distilled water after the bluing. The improved H.E staining method for the mouse bone marrow tissue section reasonably controls the time of the tissue section during staining, so that the soaking time of each step is more reasonable, the staining effect of the bone marrow tissue section is improved, the fast and efficient tissue section staining standard is achieved, and the application prospect is wide.
Description
Technical Field
The invention belongs to the technical field of detection methods, and particularly relates to an improved H.E staining method for a mouse bone marrow tissue section.
Background
Hematoxylin-eosin staining (h.emotoxylin-eosin staining), abbreviated as h.e staining, is one of the commonly used staining methods in paraffin sectioning technology. The hematoxylin staining solution is alkaline, and mainly makes the chromatin in the cell nucleus and the nucleic acid in the cytoplasm bluish; eosin is an acid dye that primarily reddens components in the cytoplasm and extracellular matrix.
In the pathological histological observation, pathological sections are often needed to be carried out on the bone marrow of a mouse so as to observe the influence of diseases or medicines on the bone marrow tissue structure, the bone marrow cell morphology, the number and the like; the H.E staining method is widely used in tissue sections, two staining solutions of alkaline hematoxylin and acidic eosin are used for staining paraffin sections, the staining quality is determined by the staining steps and the time of soaking in the staining solutions, the clarity degree in later section observation is greatly influenced, and the quality of the tissue sections is determined.
When H.E dyeing is carried out, operators should pay close attention to reagent concentration change and soaking time, and reasonably control temperature and humidity environments according to actual operation requirements, so that the reasonability, standardization and scientificity of each operation are ensured. The tissue structure of the section is complex, and a tester can control the dyeing time well in the dyeing process, and simultaneously ensure that the dyeing agent can be fully combined with the tissue, so that the tissue can be thoroughly dyed.
Standard h.e staining comprises the following steps:
(20) neutral gum sealing sheet: dipping neutral gum with a glass rod, dripping the neutral gum on the edge of the tissue on the glass slide, clamping the edge of the glass slide with forceps, slightly covering the tissue with a cover glass, and allowing the neutral gum to freely diffuse under the cover glass to cover the tissue.
However, the hematoxylin soaking staining time and the flowing water washing time after staining, and the eosin soaking staining time and the ethanol washing time after staining are difficult to control, so that too long and too short result in great influence on cell staining, the cell staining effect is poor, observation of tissue slices is not facilitated, and high-concentration alcohol can cause decalcified and softened bone tissues to shrink and harden.
Disclosure of Invention
The invention provides an improved H.E staining method for mouse bone marrow tissue sections, which aims to solve one or more technical problems in the prior art and at least provide a beneficial selection or creation condition.
In order to overcome the technical problems, the technical scheme adopted by the invention is as follows:
an improved H.E staining method for mouse bone marrow tissue sections, wherein sodium bicarbonate solution is used for treatment.
As a further improvement of the above protocol, the mouse bone marrow tissue section h.e staining method is performed by soaking with 2% sodium bicarbonate solution, and then rinsing with running water.
As a further improvement of the scheme, 50% -100% alcohol is also used in the H.E staining method of the mouse bone marrow tissue section.
As a further improvement of the scheme, 70% -100% alcohol is also used in the H.E staining method of the mouse bone marrow tissue section.
As a further improvement of the above protocol, the h.e staining method of mouse bone marrow tissue section comprises the following steps:
(19) neutral gum sealing sheet: dipping neutral gum with a glass rod, dripping the neutral gum on the edge of the tissue on the glass slide, clamping the edge of the glass slide with forceps, slightly covering the tissue with a cover glass, and allowing the neutral gum to freely diffuse under the cover glass to cover the tissue.
The invention has the beneficial effects that: the invention provides an improved H.E staining method for mouse marrow tissue sections, which is characterized in that paraffin is fully dissolved by properly increasing the time length of a prophase xylene dewaxing transparent step, so that staining solution fully enters tissues during staining; meanwhile, the concentration gradient of the alcohol and the time for soaking and dehydrating the high-concentration alcohol are reduced, and the softened bone tissue can be effectively prevented from shrinking and hardening to influence the slicing quality; the slice is blued by using 2% sodium bicarbonate in alkalescent solution, so that the blueing efficiency and effect are improved, and the cost is reduced; and the dyeing effect of the eosin solution can be improved by washing with distilled water after the bluing. The improved H.E staining method for the mouse bone marrow tissue section reasonably controls the time of the tissue section during staining, so that the soaking time of each step is more reasonable, the staining effect of the bone marrow tissue section is improved, the fast and efficient tissue section staining standard is achieved, and the method is favorable for observing the change of bone marrow cells and analyzing the disease development by the later stage. The dyeing method has the advantages of lower cost, less material consumption, enhanced dyeing effect, rapidness, reasonability, higher efficiency and wide application prospect.
Drawings
FIG. 1 is a staining diagram of a bone marrow section of a mouse stained by a modified H.E staining method obtained in example 1, which is magnified 100 times;
fig. 2 is a graph showing the staining of a bone marrow section of a mouse stained by a conventional h.e staining method according to comparative example 1, which is magnified 100-fold.
Detailed Description
The present invention is specifically described below with reference to examples in order to facilitate understanding of the present invention by those skilled in the art. It should be particularly noted that the examples are given solely for the purpose of illustration and are not to be construed as limitations on the scope of the invention, as non-essential improvements and modifications to the invention may occur to those skilled in the art, which fall within the scope of the invention as defined by the appended claims. Meanwhile, the raw materials mentioned below are not specified in detail and are all commercially available products; the process steps or extraction methods not mentioned in detail are all process steps or extraction methods known to the person skilled in the art.
Example 1
And dyeing the paraffin wax sample of the bursa of Fabricius by adopting a modified H.E dyeing method. After the sample is sliced, the sample is dyed according to the following steps:
(19) neutral gum sealing sheet: dipping neutral gum with a glass rod, dripping the neutral gum on the edge of the tissue on the glass slide, clamping the edge of the glass slide with forceps, slightly covering the tissue with a cover glass, and allowing the neutral gum to freely diffuse under the cover glass to cover the tissue.
The staining piece shown in FIG. 1 was obtained, and it can be seen from FIG. 1 that the staining degree of the tissue was deep, the contrast was significant, and the staining effect was good.
Comparative example 1
The same paraffin samples of bursa of Fabricius as in example 1 were taken for standard H.E staining: the method specifically comprises the following steps:
(20) neutral gum sealing sheet: dipping neutral gum with a glass rod, dripping the neutral gum on the edge of the tissue on the glass slide, clamping the edge of the glass slide with forceps, slightly covering the tissue with a cover glass, and allowing the neutral gum to freely diffuse under the cover glass to cover the tissue.
A dyed sheet as shown in FIG. 2 was obtained. As can be seen from FIG. 2, the staining of the tissue is light, the contrast is poor, and the staining effect ratio is not ideal.
It will be obvious to those skilled in the art that many simple derivations or substitutions can be made without inventive effort without departing from the inventive concept. Therefore, simple modifications to the present invention by those skilled in the art according to the present disclosure should be within the scope of the present invention. The above embodiments are preferred embodiments of the present invention, and all similar processes and equivalent variations to those of the present invention should fall within the scope of the present invention.
Claims (5)
1. An improved H.E staining method for mouse bone marrow tissue sections, which is characterized in that sodium bicarbonate solution is used for processing.
2. The method for H.E staining of mouse bone marrow tissue section according to claim 1, wherein the mouse bone marrow tissue section is first soaked in 2% sodium bicarbonate solution and then washed with running water.
3. The method for H.E staining of mouse bone marrow tissue section according to claim 1 or 2, wherein 50-100% alcohol is further used in the method for H.E staining of mouse bone marrow tissue section.
4. The method for H.E staining of mouse bone marrow tissue section according to claim 3, wherein 70-100% alcohol is further used in the method for H.E staining of mouse bone marrow tissue section.
5. The method for H.E staining of mouse bone marrow tissue sections according to claim 4, comprising the steps of:
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| CN202010782679.XA CN112067409A (en) | 2020-08-06 | 2020-08-06 | Improved H.E staining method for mouse bone marrow tissue section |
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| CN202010782679.XA CN112067409A (en) | 2020-08-06 | 2020-08-06 | Improved H.E staining method for mouse bone marrow tissue section |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090246824A1 (en) * | 2008-03-27 | 2009-10-01 | Richard-Allan Scientific Company | Methods for Integrated Tissue Processing and Staining |
| CN105067412A (en) * | 2015-08-10 | 2015-11-18 | 长春瑞克医疗科技有限公司 | Vaginal secretion staining fluid, and preparation method and staining method thereof |
| CN110686957A (en) * | 2019-10-11 | 2020-01-14 | 佛山科学技术学院 | Improved HE dyeing method |
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2020
- 2020-08-06 CN CN202010782679.XA patent/CN112067409A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090246824A1 (en) * | 2008-03-27 | 2009-10-01 | Richard-Allan Scientific Company | Methods for Integrated Tissue Processing and Staining |
| CN105067412A (en) * | 2015-08-10 | 2015-11-18 | 长春瑞克医疗科技有限公司 | Vaginal secretion staining fluid, and preparation method and staining method thereof |
| CN110686957A (en) * | 2019-10-11 | 2020-01-14 | 佛山科学技术学院 | Improved HE dyeing method |
Non-Patent Citations (1)
| Title |
|---|
| 方福德: "现代医学实验技巧全书 上", 北京医科大学 中国协和医科大学联合出版社国协和医科大学联合出版社国协和医科大学联合出版社, pages: 28 - 29 * |
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