Enhanced anti-fluorescence quenching mounting medium and preparation method and mounting method thereof

Abstract

The invention discloses an enhanced anti-fluorescence quenching mounting medium as well as a preparation method and a mounting method thereof, particularly relates to the technical field of immunofluorescence, and aims to solve the problems that a fluorescent dye is rapidly attenuated under the irradiation of strong exciting light and the like in the prior art. Every 100 mL of the mounting medium comprises 15-30 mL of an acid-base buffer solution, 10-20 mL of a viscosity modifier, 0.5-5 g of a fluorescence quenching resistant compound, 1-15 mL of a synergist and the balance of distilled water, and the fluorescence quenching resistant compound comprises triethylene diamine and ethylenediaminetetraacetic acid. The mounting medium provided by the invention can enhance the overall fluorescence brightness and maintain the stability of fluorescence under excitation light, thereby realizing more accurate quantification of fluorescence signals.

Description

Technical Field

[0001] The invention relates to an enhanced anti-fluorescence quenching sealing agent and a preparation method and a sealing method thereof, and belongs to the technical field of immunofluorescence. Background Art

[0002] Immunofluorescence in situ hybridization technique (Immuno-FISH), abbreviated as immuno-FISH, is a common labeling technique that uses the principle of antigen-antibody reaction to locate specific antigens or antibodies on test cells or chromosomes in cells. It is used to study the distribution and expression of specific protein antigens or antibodies on cells or chromosomes to achieve quantitative and localized detection. This technology has been widely reported in chromosome immunofluorescence localization analysis of model organisms such as humans, mice, zebrafish, and fruit flies.

[0003] For a long time, the quenching of fluorescent dyes, that is, the attenuation of fluorescence on cells or tissue sections after staining, has been the main problem that plagues fluorescent staining of cells or tissues. There are two main reasons for the quenching of fluorescent dyes: first, the properties of the dye itself. Fluorescence decay is an inevitable process, and the decay rate between fluorescent dyes varies greatly. The Alexa Fluor series dyes commonly seen on the market, such as 488, 555, 594 and 647, not only have increased brightness compared to traditional dyes, but also have a significantly slower decay rate. Second, during microscopic photography, the excitation light of the microscope, especially the laser of the confocal microscope, will accelerate the quenching rate of fluorescent dyes. This is very obvious for proteins with very low expression abundance, because the low protein expression leads to a weak fluorescence signal. When taking photos with a confocal microscope, it is often necessary to increase the intensity of the laser energy output, which causes the fluorescence signal to decay rapidly.

[0004] The weakening or quenching of fluorescence not only seriously affects the quality of the image (low optimal resolution) and the reliability of the experimental results. Most of the existing mounting agents on the market cannot solve the problem of fluorescence signal attenuation caused by strong excitation light or long shooting time. Even a small number of anti-fluorescence quenching mounting agents can only protect certain fluorescent dyes and cannot achieve full coverage. This problem is particularly prominent when the abundance of the target protein is too low. Therefore, it is urgent to develop an anti-fading mounting agent that can enhance the overall fluorescence brightness and slow down the attenuation of fluorescent dyes. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide an enhanced anti-fluorescence quenching sealing agent and a preparation method and a sealing method thereof, which can enhance the overall fluorescence brightness and maintain the stability of fluorescence under excitation light, thereby achieving more accurate quantification of fluorescence signals.

[0006] To achieve the above object, the present invention is implemented by adopting the following technical solutions: In a first aspect, the present invention provides an enhanced anti-fluorescence quenching sealing agent, wherein each 100 mL of the sealing agent comprises 15-30 mL of acid-base buffer, 10-20 mL of viscosity regulator, 0.5-5 g of anti-fluorescence quenching complex and 0.1-1 g of enhancer, and the rest is distilled water, wherein the anti-fluorescence quenching complex comprises triethylenediamine and ethylenediaminetetraacetic acid.

[0007] Optionally, the pH range of the acid-base buffer is 7.0-9.0, which includes one or more of phosphate buffer, citrate buffer, carbonate buffer and tris(hydroxymethyl)aminomethane.

[0008] Optionally, the acid-base buffer is a mixture of citric acid buffer and tris(hydroxymethyl)aminomethane.

[0009] Optionally, the viscosity modifier is glycerol and / or polyethylene glycol.

[0010] Optionally, the polyethylene glycol is one or more of low molecular weight polyethylene glycol, medium molecular weight polyethylene glycol and high molecular weight polyethylene glycol, wherein the molecular weight of the low molecular weight polyethylene glycol is 200, 400, 600 and 800, the molecular weight of the medium molecular weight polyethylene glycol is 1000, 1500, 2000 and 4000, and the molecular weight of the high molecular weight polyethylene glycol is 6000, 8000, 10000 and 20000.

[0011] Optionally, the mass ratio of triethylenediamine to ethylenediaminetetraacetic acid in the anti-fluorescence quenching complex is 1:3~6.

[0012] Optionally, the synergist is a solution of butylated hydroxyanisole and butylated hydroxytoluene dissolved in dimethyl sulfoxide.

[0013] In a second aspect, the present invention provides a method for preparing an enhanced anti-fluorescence quenching sealing agent, comprising: Mixing an acid-base buffer, a viscosity modifier, an anti-fluorescence quenching complex and distilled water to obtain a solution A; Solution A is mixed with an enhancer to obtain an enhanced anti-fluorescence quenching sealing medium.

[0014] In a third aspect, the present invention provides a sealing method for an enhanced anti-fluorescence quenching sealing agent, comprising: The cells or tissues are incubated with the target primary antibody and then fluorescently stained; Place cells or tissues on a glass slide and place a sealing agent on the cells or tissues. Apply sealant evenly on the bottom surface of the coverslip to seal all sides of the coverslip and place it on the slide to complete the mounting.

[0015] Furthermore, before covering with the coverslip, a counterstaining operation is also included, wherein the counterstaining operation includes adding a nuclear fluorescent dye into the sealing medium, wherein the nuclear fluorescent dye is 4′,6-diamidino-2-phenylindole, and the concentration range of the nuclear fluorescent dye is 0.5-10 μg / mL.

[0016] Beneficial Effects

[0017] The anti-fluorescence quenching sealing agent provided by the present invention can strongly stabilize the fluorescence characteristics in the cell or tissue sample, and greatly improve the sensitivity of capturing the fluorescence signal, maintain the fluorescence dyeing sample for a long time, effectively solve the light stability problem of the fluorescent dye in the cell or tissue sample, and greatly improve the sensitivity of the captured fluorescence signal, thereby providing a guarantee for achieving more accurate quantification of the fluorescence signal, and the anti-quenching agent of the present invention can effectively slow down the quenching degree of the fluorescent dye under the irradiation of strong excitation light; The composite formula of the anti-fluorescence quenching sealing agent provided by the present invention is unique, wherein triethylenediamine has the effect of slowing down fluorescence quenching, and plays a catalytic role with polyethylene glycol substances in the reagent; butylated hydroxyanisole and dibutylated hydroxytoluene are highly effective antioxidants with little toxic side effects, which are beneficial to the long-term storage of reagents and glass slides; The preparation method of the anti-fluorescence quenching sealing agent provided by the present invention is simple, convenient, and highly economical, and can be widely used in the protection of fluorescent signals of cells. The sealing agent of the present invention is easy to use when implementing sealing, and imaging and photography can be performed after sealing. By adding a cell nucleus fluorescent dye before sealing, sealing and counterstaining can be completed in one step, omitting the nuclear staining step, saving time and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the expression effect of the anti-fluorescence quenching mounting agent of Example 1 of the present invention on the Ki67 protein in the mouse pancreatic cancer tissue section after fluorescence staining under the corresponding excitation light at 0min, 5min, 10min and 15min respectively; Figure 2 It is a schematic diagram of the expression effect of the anti-fluorescence quenching mounting agent of Example 2 of the present invention on the Ki67 protein in the mouse pancreatic cancer tissue section after fluorescence staining under the corresponding excitation light at 0min, 5min, 10min and 15min respectively; Figure 3It is a schematic diagram of the expression effect of the anti-fluorescence quenching mounting agent of Example 3 of the present invention on nuclear staining of Ki67 protein in mouse pancreatic cancer tissue sections after fluorescence staining under corresponding excitation light at 0min, 5min, 10min, 15min and 15min later; Figure 4 It is a schematic diagram of the expression effect of the anti-fluorescence quenching mounting agent of Example 4 of the present invention on nuclear staining of Ki67 protein in mouse pancreatic cancer tissue sections after fluorescence staining under corresponding excitation light at 0min, 5min, 10min, 15min and 15min later; Figure 5 It is a schematic diagram of the expression effect of the anti-fluorescence quenching mounting agent of Example 5 of the present invention on the Ki67 protein in the mouse pancreatic cancer tissue section after fluorescence staining under the corresponding excitation light at 0min, 5min, 10min and 15min respectively; Figure 6 It is a schematic diagram of the expression effect of the anti-fluorescence quenching mounting agent of Example 6 of the present invention on the Ki67 protein in the mouse pancreatic cancer tissue section after fluorescence staining under the corresponding excitation light at 0min, 5min, 10min and 15min respectively; Figure 7 This is a schematic diagram of the expression effect of the anti-fluorescence quenching mounting agent of comparative example 1 of the present invention on the Ki67 protein in the mouse pancreatic cancer tissue section after fluorescence staining under the corresponding excitation light at 0min, 5min, 10min and 15min respectively. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0020] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article.

[0021] For the purpose of this specification and the appended claims, unless otherwise stated, all numbers expressing amounts, percentages or ratios and other numerical values ​​used in this specification and the appended claims are understood to be modified in all cases by the term "about". In addition, all ranges disclosed herein are inclusive and independently combinable.

[0022] The invention provides an enhanced anti-fluorescence quenching sealing agent. Each 100 mL of the sealing agent comprises 15-30 mL of an acid-base buffer solution, 10-20 mL of a viscosity regulator, 0.5-5 g of an anti-fluorescence quenching complex and 1-15 mL of a synergist, and the remainder is distilled water.

[0023] The acid-base buffer is selected from one or more of a phosphate buffer, a citric acid buffer, a carbonate buffer and a tris buffer. The pH of the acid-base buffer is 7.0 to 9.0, preferably 7.5.

[0024] Viscosity modifiers are glycerol and / or polyethylene glycol.

[0025] The anti-fluorescence quenching complex is a mixture of triethylenediamine and ethylenediaminetetraacetic acid, wherein the mass ratio of triethylenediamine to ethylenediaminetetraacetic acid is 1:4-6, preferably 1:4.

[0026] The synergist is a solution of butylated hydroxyanisole and butylated hydroxytoluene dissolved in dimethyl sulfoxide, and the dissolved solution accounts for 1-15% of the total volume of the sealing agent, preferably 5%.

[0027] The present invention also provides a method for preparing an enhanced anti-fluorescence quenching sealing agent, comprising the following steps: Mixing an acid-base buffer, a viscosity modifier, an anti-fluorescence quenching complex and distilled water to obtain a solution A; Solution A is mixed with an enhancer to obtain an enhanced anti-fluorescence quenching sealing medium.

[0028] The preparation method is simple and convenient, and can be widely used in the protection of fluorescent signals of cells.

[0029] The present invention also provides a sealing method for an enhanced anti-fluorescence quenching sealing agent, comprising the following steps: After the cells / tissues are incubated and bound with the target first antibody, the cells / tissues are fluorescently stained and labeled with a fluorescent second antibody to obtain fluorescently stained and labeled cells / tissues; Add the fluorescently stained cells / tissues onto a glass slide, and then add a mounting medium onto the cells / tissues; Apply sealant evenly on the bottom surface of the coverslip to seal all sides of the coverslip and place it on the slide to complete the mounting.

[0030] Furthermore, if it is necessary to counterstain the DNA in the cell nucleus, 4′,6-diamidino-2-phenylindole (DAPI) can be added to the mounting medium to complete the mounting and counterstaining in one step, eliminating the nuclear staining step, saving time and cost. The concentration of 4′,6-diamidino-2-phenylindole in the mounting medium is 0.5~10μg / mL.

[0031] The technical solution of the present invention will be described in detail below through specific embodiments.

[0032] Example 1

[0033] The embodiment of the present invention provides a method for preparing an enhanced anti-fluorescence quenching sealing agent, comprising the following steps: In a clean beaker, add 100 mL of distilled water and 10 g of citric acid, and adjust the pH to 7.5 to obtain the prepared citric acid buffer. In another beaker, add 100 mL of distilled water and 1.21 g of tris(hydroxymethyl)aminomethane, and adjust the pH to 7.5.

[0034] The citric acid buffer and the tris-hydroxymethylaminomethane hydrochloride (Tris-HCL) buffer were mixed at a volume ratio of 1:1, and mixed thoroughly for 20 minutes to obtain a mixed solution I. The mixed solution I was stored at 4°C for later use.

[0035] Take 30 mL of mixed solution I and glycerol in a volume ratio of 3:1, mix them thoroughly, and then add 1 g of triethylenediamine and 3 g of ethylenediaminetetraacetic acid in sequence to obtain solution A.

[0036] Add 0.5 g of butylated hydroxyanisole (BHA) and 0.5 g of butylated hydroxytoluene (BHT) into 10 mL of dimethyl sulfoxide and fully dissolve to obtain a mixed solution II. Divide the mixed solution II into small tubes, each with a volume of 2 mL, and store at -20°C for later use.

[0037] Solution A was fully mixed with a small tube of mixed solution II, and the volume was adjusted to 100 mL with distilled water to prepare an enhanced anti-fluorescence quenching sealing agent, which was recorded as A1 and stored in a brown or black reagent bottle at 4-8°C.

[0038] Example 2

[0039] The embodiment of the present invention provides a method for preparing an enhanced anti-fluorescence quenching sealing agent, comprising the following steps: In a clean beaker, add 100 mL of distilled water, add 10 g of citric acid, and adjust the pH to 7.5 to obtain the prepared citric acid buffer. In another beaker, add 100 mL of distilled water, add 1.84 g of potassium dihydrogen phosphate, and adjust the pH to 7.5.

[0040] The citrate buffer and the phosphate buffer were mixed in a volume ratio of 1:1, and mixed thoroughly for 20 minutes to obtain a mixed solution I, which was stored at 4°C for later use.

[0041] Take 30 mL of mixed solution I and 5 g of polyethylene glycol 200 (PEG200), mix them thoroughly, and then add 1 g of triethylenediamine and 3 g of ethylenediaminetetraacetic acid at a time to obtain solution A.

[0042] Add 0.5 g of butylated hydroxyanisole (BHA) and 0.5 g of butylated hydroxytoluene (BHT) into 10 mL of dimethyl sulfoxide and fully dissolve to obtain a mixed solution II. Divide the mixed solution II into small tubes, with a volume of 2 ml in each small tube, and store at -20°C for future use.

[0043] Solution A and mixed solution II were fully mixed, and the volume was adjusted to 100 mL with distilled water to prepare an enhanced anti-fluorescence quenching sealing agent, which was recorded as A2 and stored in a brown or black reagent bottle at 4-8°C.

[0044] Example 3

[0045] 4′,6-diamidino-2-phenylindole (DAPI) was added to the solution prepared in Example 1 at a concentration of 5 μg / mL, mixed and stored at room temperature, thereby preparing a DAPI-containing mounting medium, which was designated as A3.

[0046] Example 4

[0047] 4′,6-diamidino-2-phenylindole (DAPI) was added to the solution prepared in Example 2 at a concentration of 5 μg / mL, mixed and stored at room temperature, thereby preparing a DAPI-containing mounting medium, which was designated as A4.

[0048] Example 5

[0049] The only difference between this example and Example 1 is that "take 30 mL of mixed solution I and mix it with glycerol in a volume ratio of 3:1" is changed to "take 30 mL of mixed solution I and mix it with 5 g of polyethylene glycol 200 (PEG200)", and an enhanced anti-fluorescence quenching sealing agent is prepared, which is recorded as A5.

[0050] Example 6

[0051] The difference between this embodiment and embodiment 2 is that "1 g triethylenediamine and 3 g ethylenediaminetetraacetic acid" is changed to "3 g triethylenediamine and 1 g ethylenediaminetetraacetic acid", and an enhanced anti-fluorescence quenching sealing agent is prepared, which is recorded as A6.

[0052] Comparative Example 1 This comparative example provides an existing commercial anti-fluorescence quenching sealing agent, which is produced by Biotime Biotechnology Co., Ltd.

[0053] The performance of the sealing agents of Examples 1 to 6 and Comparative Example 1 is analyzed below.

[0054] For Examples 1 to 6: Mouse pancreatic tumor tissue sections were used, and according to conventional immunofluorescence experimental methods, rabbit anti-Ki67 antibody was selected as the primary antibody and anti-rabbit FITC was selected as the secondary antibody.

[0055] After the experiment, drop mounting media A1 to A6 onto the slide containing the sample (the amount of mounting media used should be sufficient to cover the specimen area on the cover glass, slide or chamber slide).

[0056] Use the FITC channel of an upright fluorescence microscope to illuminate the positive area for 0 min, 5 min, 10 min, and 15 min, and take pictures. The results are as follows Figure 1 shown.

[0057] from Figure 1 to Figure 6 It can be seen from the image results that the sealing agents A1-A6 protected the stability of the fluorescence signal at 0 min, 5 min, 10 min and 15 min after immunofluorescence staining, indicating that the sealing agents A1-A6 of Examples 1-6 can strongly stabilize the fluorescence characteristics in the cell samples and maintain the fluorescence staining sample time.

[0058] For comparative example 1: Mouse pancreatic tumor tissue sections were used, and according to conventional immunofluorescence experimental methods, rabbit anti-Ki67 antibody was selected as the primary antibody and anti-rabbit FITC was selected as the secondary antibody.

[0059] At the end of the experiment, drop a commercially available mounting medium onto the coverslip containing the specimen (the amount of mounting medium should be enough to cover the specimen area on the coverslip, slide, or chamber slide).

[0060] Use the FITC channel of an upright fluorescence microscope to illuminate the positive area for 0 min, 5 min, 10 min, and 15 min, and take pictures. The results are as follows Figure 7 shown.

[0061] from Figure 7 From the image results, it can be seen that the fluorescence of the commercial sealing agent gradually quenched until it was almost completely quenched at 0min, 5min, 10min and 15min after immunofluorescence staining, indicating that the effect of slowing down fluorescence quenching is poor.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An enhanced anti-fluorescence quenching sealing agent, characterized in that: Each 100 mL of the sealing agent includes 15-30 mL of acid-base buffer, 10-20 mL of viscosity regulator, 0.5-5 g of anti-fluorescence quenching complex and 1-15 mL of enhancer, and the rest is distilled water, wherein the anti-fluorescence quenching complex includes triethylenediamine and ethylenediaminetetraacetic acid.

2. The enhanced anti-fluorescence quenching mounting medium according to claim 1, characterized in that: The pH range of the acid-base buffer is 7.0-9.0, and the acid-base buffer includes one or more of a phosphate buffer, a citric acid buffer, a carbonate buffer and tris(hydroxymethyl)aminomethane.

3. The enhanced anti-fluorescence quenching sealing agent according to claim 2, characterized in that: The acid-base buffer is a mixture of citric acid buffer and tris(hydroxymethyl)aminomethane.

4. The enhanced anti-fluorescence quenching mounting medium according to claim 1, characterized in that: The viscosity modifier is glycerol and / or polyethylene glycol.

5. The enhanced anti-fluorescence quenching mounting medium according to claim 4, characterized in that: The polyethylene glycol is one or more of low molecular weight polyethylene glycol, medium molecular weight polyethylene glycol and high molecular weight polyethylene glycol, wherein the molecular weight of the low molecular weight polyethylene glycol is 200, 400, 600 and 800, the molecular weight of the medium molecular weight polyethylene glycol is 1000, 1500, 2000 and 4000, and the molecular weight of the high molecular weight polyethylene glycol is 6000, 8000, 10000 and 20000.

6. The enhanced anti-fluorescence quenching mounting medium according to claim 1, characterized in that: The mass ratio of triethylenediamine to ethylenediaminetetraacetic acid in the anti-fluorescence quenching complex is 1:3-6.

7. The enhanced anti-fluorescence quenching mounting medium according to claim 1, characterized in that: The synergist is a solution of butylated hydroxyanisole and butylated hydroxytoluene dissolved in dimethyl sulfoxide.

8. A method for preparing the enhanced anti-fluorescence quenching sealing agent according to any one of claims 1 to 7, characterized in that: include: Mixing an acid-base buffer, a viscosity modifier, an anti-fluorescence quenching complex and distilled water to obtain a solution A; Solution A is mixed with an enhancer to obtain an enhanced anti-fluorescence quenching sealing medium.

9. A method for sealing the enhanced anti-fluorescence quenching sealing agent according to any one of claims 1 to 7, characterized in that: include: After the cells or tissues are incubated and bound with the target first antibody, the cells or tissues are fluorescently stained and labeled with a fluorescent second antibody to obtain fluorescently stained cells / tissues; Drop the fluorescently stained cells / tissues on a glass slide, and drop a mounting medium on the cells or tissues; Apply sealant evenly on the lower surface of the coverslip to seal all sides of the coverslip and place it on the slide to complete the mounting.

10. The sealing method of the enhanced anti-fluorescence quenching sealing agent according to claim 9, characterized in that: Before covering with the coverslip, a counterstaining operation is also included, wherein the counterstaining operation includes adding a cell nucleus fluorescent dye into the sealing medium, wherein the cell nucleus fluorescent dye is 4',6-diamidino-2-phenylindole, and the concentration range of the cell nucleus fluorescent dye is 0.5-10 μg / mL.

Images