CN107063814A - Lead citrate staining solution and its compound method and application - Google Patents

Abstract

The invention relates to a lead citrate staining solution and its preparation method and application. The lead citrate staining solution includes a solvent, lead citrate with a concentration of 30-40 g/L and sodium hydroxide with a concentration of 0.5-0.8 g/L in the solvent; the solvent is a mixed solution of alcohol and water , wherein, the volume concentration of the alcohol is 25-33%. The preparation method of the lead citrate staining solution is simple, the staining effect is good, the presentation of the ultrastructure is beneficial, the storage is stable, the storage period is long, and the pollution is small and the cost is low.

Description

Lead citrate staining solution and its preparation method and application

technical field

The invention relates to a dyeing solution, in particular to a lead citrate dyeing solution and a preparation method and application thereof.

Background technique

For ultra-thin sections of biological samples, the image contrast comes from the scattering ability of the sample for the electron beam, and its scattering ability depends on the atomic composition: the higher the atomic number, the higher the electron density, the stronger the ability to scatter electrons, and it appears black under the electron microscope ; The lower the atomic number, the lower the electron density, the weaker the ability to scatter electrons, and it appears white under the electron microscope. However, biological samples are mainly composed of elements with low atomic numbers such as C, H, O, N, P, and S. Unstained ultra-thin sections have very weak contrast, especially for sections used for medical diagnosis. The poor contrast effect will lead to The structure is not clear, which seriously affects the diagnostic results of pathologists.

The commonly used staining solutions for ultrathin sections include uranyl acetate staining solution and lead citrate staining solution. Among them, uranyl acetate: also known as uranyl acetate, it mainly improves the contrast of nucleic acid, protein and connective tissue fibers, and has a poor staining effect on membranes. Lead citrate: It has a high density and has a wide range of affinity effects on various tissue structures. It is especially good for improving the contrast of cell membrane systems and lipid substances. It has a better staining effect on glycogen that cannot be stained by osmic acid. Due to the different staining characteristics of uranium and lead, double staining is commonly used in slides. That is, after staining with uranyl acetate, and then staining with lead citrate, they complement each other to obtain a better staining effect.

However, the preparation of the current lead citrate staining solution is relatively difficult, and it is easy to combine with carbon dioxide in the air during storage and staining to form lead carbonate particles to contaminate the slices, thereby affecting electron microscope observation, and with the increase of staining time and number of slices will increase the possibility of contamination. The steps of the lead citrate staining solution prepared by the laboratory are relatively complicated, and it is difficult to make the solution transparent during the preparation process, and the staining solution is unstable. After preparation, it is stored for a period of time (such as after two weeks), and precipitation is prone to occur, resulting in the inability of the staining solution. use. Although the commercialized lead citrate staining solution has a long storage period, the preparation method is very complicated. If operations such as darkening, airtight stirring, filter ball filtration, nitrogen filling, and melting sealing are required, it is difficult for general laboratories to prepare. In addition, both laboratory self-prepared and commercialized lead citrate staining solutions have problems such as unstable staining effect, prone to precipitation, and unsatisfactory staining effect.

In addition, in the general knowledge of those skilled in the art, water is recognized as the preferred solvent for preparing the dyeing solution, because it can dissolve the solute (lead citrate) very well, and will not react abnormally with lead citrate , the solution with water as solvent is generally considered to have better stability than organic solvents, and because the lead citrate dye solution has a requirement for pH value, it is also easy to adjust the pH value with water as solvent. Therefore, no matter the laboratory self-made or commercialized lead citrate staining solution, the aqueous solution is used as the solvent. However, the resulting staining solution has a high surface tension, and it is easy to form small air bubbles with the slices, resulting in diffuse pollution, and it is easy to form small air bubbles on the surface of the ultra-thin slices during staining, and it is not easy to combine well with the ultra-thin slices. , affecting the sharpness of ultrathin sections. Moreover, the dyeing solution has weak permeability. After the staining is completed, the ultra-thin sections are not easy to rinse, and the lead citrate staining solution will be more or less residual, causing pollution and resulting in poor staining quality of the ultra-thin sections. Lots of water. The pollution and cost of the above-mentioned lead citrate staining solution, as well as the impact on the quality of ultrathin sections have been ignored by those skilled in the art.

Contents of the invention

Based on this, it is necessary to provide a lead citrate staining solution with simple preparation method, good staining effect, favorable ultrastructure presentation, stable storage, long storage period, little pollution and low cost.

A lead citrate staining solution, comprising a solvent, and lead citrate with a concentration of 30 to 40 g/L and sodium hydroxide with a concentration of 0.5 to 0.8 g/L in the solvent;

The solvent is a mixed solution of alcohol and water, wherein the volume concentration of the alcohol is 25-33%.

In the prior art, no matter the lead citrate staining solution prepared by the laboratory or commercialized, the aqueous solution is used as the solvent. The lead citrate staining solution of the present invention is a breakthrough attempt to add alcohol as a solvent in the lead citrate staining solution to reduce the surface tension of the staining solution. At the same time, it is found through research that if the concentration of alcohol is too large, it is difficult to make a clear solution, and it will affect the stability of the lead citrate staining solution; if the concentration of alcohol is too small, it is difficult to reduce the surface tension of the staining solution.

Finally, the present invention cooperates with lead citrate and sodium hydroxide with the alcohol-containing solvent of specific volume concentration, not only improves the concentration of active ingredient lead citrate in the lead citrate dyeing solution, strengthens the dyeing effect of the dyeing solution, shortens the dyeing time , and in this case also can guarantee the stability of the lead citrate staining solution that makes, and storage period is long. At the same time, the surface tension of the dyeing solution is reduced, so that the prepared ultra-thin sections fit tightly, the permeability of the dyeing solution is improved, the dyeing effect is good, and it is easy to rinse, saving the amount of ultrapure water for washing, and avoiding lead pollution caused by the residue of the dyeing solution.

In one embodiment, the volume concentration of the alcohol in the solvent is 28-32%.

In one embodiment, the alcohol is methanol and/or ethanol. In the lead citrate staining solution of the present invention, better stability can be obtained by using methanol and/or ethanol as the alcohol.

In one embodiment, the alcohol is ethanol. Ethanol is preferably used, which can reduce the toxicity of the lead citrate staining solution while ensuring the stability.

In one embodiment, in the solvent, the concentration of the lead citrate is 34-36 g/L, and the concentration of the sodium hydroxide is 0.6-0.7 g/L.

The present invention also provides the preparation method of described lead citrate staining liquid, comprises the steps:

(1) Mix the lead citrate, alcohol, and water equal to the volume of the alcohol, and use ultrasonic vibration to obtain a premix;

(2) Add the sodium hydroxide and the remaining water into the premix, and then perform ultrasonic vibration to obtain the lead citrate staining solution.

The preparation method of the lead citrate staining solution of the present invention is simple in operation, easy to control, and can be quickly prepared in a short time. Wherein, in step (1), earlier adopt the water of equal volume with alcohol, can suitably dilute the concentration of alcohol, lead citrate can be preferably dissolved.

In one of the embodiments, the ultrasonic oscillation frequency is 35-45KHz. The use of ultrasonic vibration at a specific frequency can shorten the preparation time of the staining solution and reduce the adverse effects of CO ₂ on the staining solution.

In one embodiment, in step (1), the time of the ultrasonic vibration is 2-8 minutes.

In one embodiment, in step (2), the ultrasonic vibration time is 5-10 minutes.

The invention also provides the application of the lead citrate staining solution in ultrathin sections.

Compared with the prior art, the present invention has the following beneficial effects:

The lead citrate staining solution described in the present invention breaks the traditional limitation that the formulations of the current lead citrate staining solutions are aqueous solutions, and develops a lead citrate staining solution that can use alcohol as a solvent and is stable and has better dyeing effects . It has the following advantages:

1. Good staining effect: alcohol is added as a solvent in the staining solution, and the content of the active ingredient lead citrate in the staining solution is increased, so that it can penetrate into the slice better, and is more conducive to combining with the tissue structure, thereby producing obvious Contrast, so that the section can present a clearer ultrastructure under the electron microscope;

2. Stable storage and long storage period;

3. The surface tension is small, no small bubbles will be formed with the slice, and there is less pollution;

4. After the staining is completed, the slices are easy to rinse. If you use this staining solution to dye 100 slices at a time, you only need 300mL of ultra-pure water, and the washing time is about 1 minute. lead to lead pollution in slices, and can also reduce the production cost of slices;

5. The amount of staining solution is less: due to the small surface tension of the staining solution, it is easier to spread evenly in the slice, and at least 30% of the staining solution can be saved in the same amount of section staining;

6. The preparation method is simple, and the preparation can be completed in a short time;

7. It can be directly applied to dyeing without centrifugation and other operations.

Description of drawings

Fig. 1 is the ultrathin section electron micrograph that adopts the lead citrate staining solution of embodiment 1 preparation to make;

Fig. 2 is the ultrathin section electron micrograph that adopts the lead citrate staining solution of embodiment 2 preparation to make;

Fig. 3 is the ultrathin section electron micrograph that adopts the lead citrate staining solution of embodiment 3 preparation to make;

Fig. 4 is the ultrathin section electron micrograph that adopts the lead citrate staining solution of comparative example 3 preparation to make;

FIG. 5 is an electron micrograph of an ultrathin section prepared by using the lead citrate staining solution prepared in Comparative Example 4.

detailed description

The lead citrate staining solution of the present invention and its preparation method and application are described in further detail below in conjunction with specific examples.

In the embodiment of the present invention, the water is ultrapure water, which is specifically obtained by distilling the first-grade water from a Milli-Q Gradient pure water machine through a household distilled water machine.

Raw materials, attached materials and equipment are shown in Table 1:

Table 1

Example 1

In this embodiment, a lead citrate staining solution comprises 100 mL of solvent, 3.5 g of lead citrate and 0.064 g of sodium hydroxide dissolved in the solvent.

The solvent is a mixture of ethanol and ultrapure water, wherein the volume concentration of ethanol is 30%, ie 30mL.

The preparation method of above-mentioned lead citrate staining liquid, comprises the steps:

Take 3.5g of lead citrate, add 30mL of ethanol and 30mL of ultrapure water, mix and carry out ultrasonic vibration for 5min at a frequency of 40KHz to obtain a premix;

Take an appropriate amount of ultrapure water to dissolve 0.064g of sodium hydroxide to make 16mL of 1N sodium hydroxide solution, and add the sodium hydroxide solution to the above premix, then dilute to 100mL with ultrapure water, and then carry out Ultrasonic vibration for 5 to 10 minutes to completely dissolve the solid, and the solution is clear to obtain the lead citrate staining solution, which is divided into 5 or 10 ml syringes, sealed and refrigerated for storage.

The lead citrate staining solution was used for staining without any lead pollution, with clear ultrastructure and good contrast (Figure 1). When cleaning, 100 slices are rinsed with 300mL ultrapure water for 1min.

Example 2

In this embodiment, a lead citrate staining solution comprises 100 mL of solvent, 3 g of lead citrate and 0.064 g of sodium hydroxide dissolved in the solvent.

The solvent is a mixture of ethanol and ultrapure water, wherein the volume concentration of ethanol is 30%, ie 30mL.

The preparation method of above-mentioned lead citrate staining liquid is similar to embodiment 1.

Adopt this lead citrate dyeing solution to carry out dyeing, ultrastructure is clear, and contrast is also good (Fig. 2), want to obtain the dyeing effect of embodiment 1, need to prolong the dyeing time appropriately, but will increase the probability of pollution thus. When cleaning, 100 slices are rinsed with 300mL ultrapure water for 1min.

Example 3

In this embodiment, a lead citrate staining solution comprises 100 mL of solvent, 4 g of lead citrate and 0.064 g of sodium hydroxide dissolved in the solvent.

The solvent is a mixture of ethanol and ultrapure water, wherein the volume concentration of ethanol is 30%, ie 30mL.

The preparation method of above-mentioned lead citrate staining liquid is similar to embodiment 1.

Using the lead citrate staining solution for staining, the ultrastructure is clear and the contrast is good (Figure 3). However, due to the increased concentration of lead citrate, the probability of CO ₂ combining with it to produce precipitation increases. When cleaning, 100 slices are rinsed with 400mL ultrapure water for 1min30S.

Example 4

In this embodiment, a lead citrate staining solution comprises 100 mL of solvent, 3.5 g of lead citrate and 0.064 g of sodium hydroxide dissolved in the solvent.

The solvent is a mixture of ethanol and ultrapure water, wherein the volume concentration of ethanol is 25%, ie 25mL.

The preparation method of above-mentioned lead citrate staining liquid is similar to embodiment 1.

Using this lead citrate staining solution for staining, the clarity of ultrastructure is acceptable. When cleaning, 100 slices are rinsed with 400mL ultrapure water for 1min30S.

Comparative example 1

This comparative example is a lead citrate staining solution, including 100 mL of solvent, 3.5 g of lead citrate and 0.064 g of sodium hydroxide dissolved in the solvent.

The solvent is a mixture of ethanol and ultrapure water, wherein the volume concentration of ethanol is 35%, ie 35mL.

The preparation method of above-mentioned lead citrate staining liquid is similar to embodiment 1.

The lead citrate dyeing solution is used for dyeing, and the organic solvent concentration is relatively high, which is not conducive to the preparation of the dye solution, and it is easy to fail in the preparation, and it is difficult to obtain a clear and pollution-free dye solution.

Comparative example 2

This comparative example is a lead citrate staining solution, including 100 mL of solvent, 3.5 g of lead citrate and 0.064 g of sodium hydroxide dissolved in the solvent.

The solvent is a mixture of ethanol and ultrapure water, wherein the volume concentration of ethanol is 40%, ie 40mL.

The preparation method of above-mentioned lead citrate staining liquid is similar to embodiment 1.

If the lead citrate dyeing solution is used for dyeing, the concentration of the organic solvent is too high, which is not conducive to the configuration of the dye solution, resulting in the failure of the configuration, which is manifested as the inability to obtain a clear and pollution-free dye solution.

Comparative example 3

This comparative example is a lead citrate staining solution, including 100 mL of solvent, 3.5 g of lead citrate and 0.064 g of sodium hydroxide dissolved in the solvent.

The solvent is a mixture of ethanol and ultrapure water, wherein the volume concentration of ethanol is 20%, ie 20mL.

The preparation method of above-mentioned lead citrate staining liquid is similar to embodiment 1.

Using this lead citrate staining solution for staining, the ultrastructure can be identified, but the clarity is not enough (Figure 4), and compared with the existing lead citrate staining solution prepared with water as a solvent, the effect correlation is not obvious enough. When cleaning, 100 slices are rinsed with 500mL ultrapure water for 2min.

Comparative example 4

This comparative example is a kind of lead citrate staining solution, including 100mL of solvent, and 2g of lead citrate and 0.064g of sodium hydroxide dissolved in the solvent.

The solvent is a mixture of ethanol and ultrapure water, wherein the volume concentration of ethanol is 30%, ie 30mL.

The preparation method of above-mentioned lead citrate staining liquid is similar to embodiment 1.

Using the lead citrate staining solution for staining, the ultrastructure can be identified, but the clarity is not enough (Figure 5). Due to the low concentration of lead citrate, in order to obtain a clear ultrastructure, it is necessary to prolong the dyeing time and increase the temperature, which greatly increases the probability of contamination. When cleaning, 100 slices are rinsed with 300mL ultrapure water for 1min.

The lead citrate staining solution of embodiment 1-4 prepares the successful inspection standard as shown in table 2.

Table 2

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (10)

1. a kind of lead citrate staining solution, it is characterised in that including solvent, and concentration in the solvent is 30~40g/ L lead citrate and concentration is 0.5~0.8g/L sodium hydroxide；

The solvent is the mixed liquor of alcohol and water, wherein, the volumetric concentration of the alcohol is 25~33%.

2. lead citrate staining solution according to claim 1, it is characterised in that in the solvent, the volume of the alcohol is dense Spend for 28~32%.

3. lead citrate staining solution according to claim 1, it is characterised in that the alcohol is methanol and/or ethanol.

4. lead citrate staining solution according to claim 3, it is characterised in that the alcohol is ethanol.

5. the lead citrate staining solution according to claim any one of 1-4, it is characterised in that described in the solvent The concentration of lead citrate is 34~36g/L, and the concentration of the sodium hydroxide is 0.6~0.7g/L.

6. the compound method of the lead citrate staining solution described in claim any one of 1-5, it is characterised in that including following step Suddenly：

(1) by the lead citrate, alcohol, and the water isometric with the alcohol is mixed, and using ultrasonic oscillation, obtains pre-composition；

(2) sodium hydroxide and remaining water are added in the pre-composition, then carries out ultrasonic oscillation, the lemon is produced Lemon lead plumbate dyeing liquor.

7. the compound method of lead citrate staining solution according to claim 6, it is characterised in that the ultrasonic oscillation Frequency is 35~45KHz.

8. the compound method of lead citrate staining solution according to claim 7, it is characterised in that in step (1), described super The time of sound wave shock is 2~8min.

9. the compound method of lead citrate staining solution according to claim 7, it is characterised in that in step (2), described super The time of sound wave shock is 5~10min.

10. application of the lead citrate staining solution described in claim any one of 1-5 in ultra-thin section.