CN1441239A - Organism tissue staining method - Google Patents
Organism tissue staining method Download PDFInfo
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- CN1441239A CN1441239A CN 02158591 CN02158591A CN1441239A CN 1441239 A CN1441239 A CN 1441239A CN 02158591 CN02158591 CN 02158591 CN 02158591 A CN02158591 A CN 02158591A CN 1441239 A CN1441239 A CN 1441239A
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Abstract
The present invention relates to organism tissue staining method with RE fluorescent complex. The staining reagent used in the present invention is solution of RE fluorescent complex, and the solution is dropped onto the organism tissue or the test sample is soaked in the solution for 0.5-3 min before flushing with distilled water. Under the exciting of ultraviolet light, the configuration and detailed structure of the stainless tissue sample may be observed in fluorescent microscope. The said method has simple operatino and short period, and the sample needs no pre-treatment and unlimited. The reagent has no or only weak toxicity and the staining process has no influence on the structure of tissue or cell. The tissue may be extracorporeal or intracorporeal.
Description
Technical field:
The present invention relates to a kind of bio-tissue colouring method, relate in particular to a kind of method of utilizing rare earth fluorescence complex to carry out bio-tissue dyeing as coloring agent.
Background technology:
After it is found that the cell that constitutes human body,, be important field of research to being the understanding of micromechanism of the biosome of representative with the human body always.Utilizing chemical substance to make cell or tissue dyeing is a kind of important research means, from dye with haematine (Hematoxylin) and Yihong (Eosin) (being called for short HE) is the immunohistochemical staining of the group dyeing of representative to labeling of monoclonal antibody, improved resolution characteristic widely, simultaneously, also widened eye-level, development along with fluorescent reagent and Fluirescence observation instrument, the fluorescence groupization, immunofluorescence dyeing, be widely used in the research field of life science with its distinctive color, and become popular research topic.Yet the defective of traditional colouring method is that step is loaded down with trivial details, time-consuming, and sample usually needs through special processing; Staining reagent toxicity is bigger in addition, generally is unsuitable for biological tissue dyeing.
Summary of the invention:
The purpose of this invention is to provide a kind of step simple, save time the rapid dyeing method that coloring agent is nontoxic or toxicity is extremely weak, and can be used for the dyeing of live body or in vitro tissue.
Bio-tissue colouring method of the present invention, its step comprises
1) on the bio-tissue sample, drips, smears, sprays or inject coloring agent solution, or the bio-tissue sample was soaked in the coloring agent solution 0.5-3 minute;
Described coloring agent solution is the rare earth fluorescence complex solution of concentration 1-50 mg/litre, and this solution solvent is selected from water, ethanol, tetrahydrofuran, chloroform, benzene or heptane or their potpourri; Rare earth element in the described rare earth fluorescence complex is selected from europium or terbium, and its part is selected from:
1. binary part, wherein a kind of part is selected from the beta-diketon class, comprises diacetone, thenyltrifluoroacetone, and another kind of part is selected from triphenylphosphine oxide, adjacent phenanthrene, and to cough up purine, pyridine, dipyridine or its substituting group be C
2-C
5The substituent of alkyl or phenyl; Or 2. pyridine, or dipyridine, perhaps its substituting group is C
2-C
5The substituent of alkyl or phenyl; Or 3. organic carboxyl acid class and substituent thereof, substituting group comprises C
2-C
5Alkyl, phenyl or acyloxy; Or the quinolinones that 4. replaces, comprise Ofloxacin, Ciprofloxacin, or orfloxacin;
Or 5. phosphine oxide class, comprise the phosphine oxide of alkylphosphine oxide, phenylphosphine oxide or its monobasic or polynary replacement.
2) clean the bio-tissue sample;
3) under ultraviolet excitation, observe the bio-tissue sample.
Organism sample of the present invention can be the biological tissue or the in vitro tissue of human body, big molecule such as nucleic acid, protein, phosphatide.
The arranging effect of following coloring agent and bio-tissue sample is better:
Coloring agent is europium-Thenoyl-triphenylphosphine oxide, and the bio-tissue sample is a phosphatide.
Coloring agent is terbium-Ciprofloxacin, and the bio-tissue sample is the mucin new-nucleo.
Coloring agent is europium-Ofloxacin, or terbium-acetylsalicylic acid, and the bio-tissue sample is a new-nucleo.
Coloring agent is terbium-acetylsalicylic acid, or europium-Ofloxacin, and the bio-tissue sample is a breast tissue.
Coloring agent is europium-Ofloxacin, or terbium-Ciprofloxacin, and the bio-tissue sample is a cancerous lung tissue.
Coloring agent is terbium-Ciprofloxacin, and the bio-tissue sample is a breast cancer tissue.
Coloring agent is terbium-Ciprofloxacin, and the bio-tissue sample is the section of salivary gland epithelial tissue.
Coloring agent is europium-orfloxacin, and the bio-tissue sample is an oral cavity inner gland bubble structure histotomy.
Rare earth fluorescence complex of the present invention has the fluorescent complex of specific staining performance, it can act on biomacromolecules such as nucleic acid, protein or phosphatide, and produce good distinctive Color, under ultraviolet excitation, use fluorescent microscope, can see the form and the CONSTRUCTED SPECIFICATION (as shown in Figure 1, 2, 3) of tissue element, and different cells is shown different coloring effects with tissue.Adopt this class complex compound to carry out biological tissue's dyeing, the result that observation post gets is with more consistent with traditional observed result of HE decoration method.Thereby can pair cell, the physiology of tissue and pathological state and structure thereof normally or unusually make judgement.
The present invention is by dripping, smear, spray or inject coloring agent solution on tissue sample, or it was soaked 0.5-3 minute in above-mentioned solution, uses the distilled water flushing sample then, and sample is painted.Under the exciting of ultraviolet light, can present green, redness, orange or blue multiple color.When two dyeing, carry out successively respectively according to above-mentioned steps.Sample after the dyeing under ultraviolet excitation, with the institutional framework of fluorescent microscope, endoscope or visual inspection dyeing, can determine to organize normally whether.
The excellent characteristics of the inventive method:
(1) the inventive method is simple to operation, and sample does not need pre-service, saves time, and is bright in luster, and clear-cut for drawing the conclusion whether biological tissue pathology takes place as early as possible, has been created reliable foundation.
(2) implement the inventive method, used biological sample is not subjected to the restriction of kind, origin, from bacterium, viral cell tissue to higher organism, coloration can both take place.
(3) dyeing course reagent avirulence or toxicity atomic a little less than, the original structure of pair cell or tissue has no effect.
(4) dyeing course is not subjected in vitro tissue or in the restriction of soma.
Except aforesaid dyeing by pair cell, biomacromolecule or biological tissue determine its whether normal, can also study the feature of physiological function, morphosis and the microstate of cell or tissue by colouring method of the present invention, simultaneously, also can be used to observe or study the physiology and the pathologic process of subcellsular level and biomacromolecule level.Because the fluorescent characteristic of agents useful for same in the inventive method, it can act on the photosensitive position of cell or tissue or relevant material, thereby produces the effect of interrupting pathologic process.In a word, the inventive method can be applicable to fields such as molecular biology, histotomy pathological study, medical verification, health and epidemic prevention.
Description of drawings:
Fig. 1 the reddish violet agglomerate occurs for phosphatide dyeing back under ultraviolet light.
Behind Fig. 2 mucin stain under ultraviolet light bright green appears.
Fig. 3 nucleic acid the city redness occurs after receiving and dyeing under ultraviolet light.
The vascular wall dyeing back of Fig. 4 breast tissue bright green occurs under ultraviolet light.
The nucleus dyeing back of Fig. 5 cancerous lung tissue bright green occurs under ultraviolet light.
Fig. 6 breast cancer surrounding tissue galandular epithelium dyeing back green fluorescence occurs under ultraviolet light.
The section of Fig. 7 (A) salivary gland epithelial tissue is observed the figure of seeing through HE dyeing with ordinary optical microscope.
(B) salivary gland epithelial tissue section is dyeed through the husky star of complex compound terbium-ring, under the exciting of ultraviolet light, and the figure that arrives with fluorescence microscope.
Fig. 8 (A) oral cavity inner gland bubble structure histotomy is observed seen figure through eosinophilic staining with ordinary optical microscope.
(B) oral cavity inner gland bubble structure histotomy under the exciting of ultraviolet light, is used fluorescence microscope, the figure of being seen through complex compound europium-orfloxacin dyeing.
Embodiment:
In order to be illustrated more clearly in the present invention, list the following example, but it there is not any restriction to scope of the present invention.
Embodiment 1
Fluorescent complex europium-thenoyltrifluoroacetone-triphenylphosphine oxide is mixed with the ethanolic solution of 5 mg/litre, it is added drop-wise on the phosphatide, after one minute, use distilled water flushing, use fluorescence microscope then, phosphatide is coloured to the purple lumps, as shown in Figure 1.
Embodiment 2
With the method for similar embodiment 1, be that the terbium-ciprofloxacin solution of 1 mg/litre makes mucin stain with concentration, the time is 3 minutes, under ultraviolet excitation, bright green fluorescence occurs, as shown in Figure 2.
Embodiment 3
With the method for similar embodiment 1, be that the terbium-Ofloxacin solution of 10 mg/litre makes new-nucleo dyeing with concentration, dyeed 3 minutes, under ultraviolet excitation, the fluorescent graphic of shiny red appears, as shown in Figure 3.
Embodiment 4
With the method for similar embodiment 1, be that the terbium-acetylsalicylic acid solution of 50 mg/litre makes the breast tissue section statining with concentration, dyeing time 0.5 minute, under ultraviolet excitation, the bright green fluorescent graphic that vascular endothelial cell presents, as shown in Figure 4.
Embodiment 5
With the method for similar embodiment 1, be that the europium-Ofloxacin solution of 30 mg/litre made the cancerous lung tissue section statining 1 minute with concentration, under ultraviolet excitation, shiny red fluorescence appears in nucleus, as shown in Figure 5.
Embodiment 6
With the method for similar embodiment 1, be that the terbium-ciprofloxacin solution of 40 mg/litre makes breast cancer tissue's section statining with concentration, dyeing time 1 minute, under ultraviolet excitation, the fluorescent graphic of the bright green that epithelial tissue presents, as shown in Figure 6.
Embodiment 7
Adopt the section of salivary gland epithelial tissue, with the dyeing of HE decoration method, observe with ordinary optical microscope, obtain the image shown in Fig. 7 (A), wherein the particle of darkviolet is a haematine crystal.And above-mentioned histotomy is with the method for similar embodiment 1, is the husky star solution-dyed of rare-earth complex terbium-ring 1.5 minutes of 20 mg/litre with concentration, under ultraviolet excitation, uses fluorescence microscope, and its nucleus is bright green, shown in Fig. 7 (B).Compare two kinds of colouring methods, the tissue morphology that it obtained is consistent.
Embodiment 8
With oral cavity inner gland bubble structure histotomy eosinophilic staining, to observe with ordinary optical microscope, the figure that is obtained is shown in Fig. 8 (A); With above-mentioned histotomy method with similar embodiment 1, be the complex compound europium-aloperidin solution dyeing 3 minutes of 5 mg/litre with concentration, under ultraviolet excitation, use fluorescence microscope, observed figure is shown in Fig. 8 (B).Wherein the part shown in the arrow is the acinus structure, compares two kinds of decoration methods, and the tubular structure in its shown tissue is consistent, just is of different shades.
Claims (10)
1, a kind of bio-tissue colouring method, its step comprises
1) on the bio-tissue sample, drips, smears, sprays or inject coloring agent solution, or the bio-tissue sample was soaked in the coloring agent solution 0.5-3 minute;
2) clean the bio-tissue sample;
3) under ultraviolet excitation, observe the bio-tissue sample;
It is characterized in that
Described coloring agent solution is the rare earth fluorescence complex solution of concentration 1-50 mg/litre, and this solution solvent is selected from water, ethanol, tetrahydrofuran, chloroform, benzene or heptane or their potpourri; Rare earth element in the described rare earth fluorescence complex is selected from europium or terbium, and its part is selected from:
1. binary part, wherein a kind of part is selected from the beta-diketon class, comprises diacetone, thenyltrifluoroacetone, and another kind of part is selected from triphenylphosphine oxide, adjacent phenanthrene, and to cough up purine, pyridine, dipyridine or its substituting group be C
2-C
5The substituent of alkyl or phenyl; Or 2. pyridine, or dipyridine, perhaps its substituting group is C
2-C
5The substituent of alkyl or phenyl; Or 3. organic carboxyl acid class and substituent thereof, substituting group comprises C
2-C
5Alkyl, phenyl or acyloxy; Or the quinolinones that 4. replaces, comprise Ofloxacin, Ciprofloxacin, or orfloxacin; Or 5. phosphine oxide class, comprise the phosphine oxide of alkylphosphine oxide, phenylphosphine oxide or its monobasic or polynary replacement.
2, bio-tissue colouring method as claimed in claim 1 is characterized in that described bio-tissue sample is selected from the biological tissue or the in vitro tissue of human body, big molecule such as nucleic acid, protein, phosphatide.
3, bio-tissue colouring method as claimed in claim 2 is characterized in that described coloring agent is europium-Thenoyl-triphenylphosphine oxide, and the bio-tissue sample is a phosphatide.
4, bio-tissue colouring method as claimed in claim 2 is characterized in that described coloring agent is terbium-Ciprofloxacin, and the bio-tissue sample is the mucin new-nucleo.
5, bio-tissue colouring method as claimed in claim 2 is characterized in that described coloring agent is europium-Ofloxacin, or terbium-acetylsalicylic acid, and the bio-tissue sample is a new-nucleo.
6, bio-tissue colouring method as claimed in claim 2, the described coloring agent of its feature is terbium-acetylsalicylic acid, europium-Ofloxacin, bio-tissue sample are breast tissue.
7, bio-tissue colouring method as claimed in claim 2 is characterized in that described coloring agent is europium-Ofloxacin, and terbium-Ciprofloxacin, bio-tissue sample are cancerous lung tissue.
8, bio-tissue colouring method as claimed in claim 2 is characterized in that described coloring agent is terbium-Ciprofloxacin, and the bio-tissue sample is a breast cancer tissue.
9, bio-tissue colouring method as claimed in claim 2 is characterized in that described coloring agent is terbium-Ciprofloxacin, and the bio-tissue sample is the section of salivary gland epithelial tissue.
10, bio-tissue colouring method as claimed in claim 2 is characterized in that described coloring agent is europium-orfloxacin, and the bio-tissue sample is an oral cavity inner gland bubble structure histotomy.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021585911A CN1193220C (en) | 2002-12-26 | 2002-12-26 | Organism tissue staining method |
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|---|---|---|---|
| CNB021585911A CN1193220C (en) | 2002-12-26 | 2002-12-26 | Organism tissue staining method |
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| CN1193220C CN1193220C (en) | 2005-03-16 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102721690A (en) * | 2012-05-10 | 2012-10-10 | 山西大学 | Chemical dyeing method for H2S in plant tissue |
| CN101228428B (en) * | 2005-05-18 | 2012-11-07 | 斯蒂尔奥尼克国际公司 | Fluorescent nanoscopy method |
| CN108709786A (en) * | 2018-02-08 | 2018-10-26 | 中国科学院化学研究所 | The dyeing and quantitative analysis method of rare earth nanometer particle in biological tissue |
-
2002
- 2002-12-26 CN CNB021585911A patent/CN1193220C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101228428B (en) * | 2005-05-18 | 2012-11-07 | 斯蒂尔奥尼克国际公司 | Fluorescent nanoscopy method |
| CN102023148B (en) * | 2005-05-18 | 2013-07-24 | 斯蒂尔奥尼克国际公司 | Fluorescent nanoscopy method |
| CN102721690A (en) * | 2012-05-10 | 2012-10-10 | 山西大学 | Chemical dyeing method for H2S in plant tissue |
| CN102721690B (en) * | 2012-05-10 | 2013-11-20 | 山西大学 | Chemical dyeing method for H2S in plant tissue |
| CN108709786A (en) * | 2018-02-08 | 2018-10-26 | 中国科学院化学研究所 | The dyeing and quantitative analysis method of rare earth nanometer particle in biological tissue |
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| Publication number | Publication date |
|---|---|
| CN1193220C (en) | 2005-03-16 |
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