JPH07146290A - Mordant for electron microscope observation - Google Patents

Abstract

PURPOSE:To achieve dyeing where image quality is totally homogeneous as in double dyeing according to acetic uranyl and lead citrate and operation is superb by using a tea leaf extract as a mordant before the double dyeing for dyeing the fiber construction in a connective tissue. CONSTITUTION:Green tea, oolong tea, and black tea are available as tea leaf extracts and the oolong tea is the optimum. The dipping time of a living body sample thin piece to a mordant for electron microscope is approximately 5-15 minutes without requiring any strict control. The double dyeing of the dipped living body sample thin piece is performed, for example, by a method utilizing acetic uranyl and lead citrate. By using the mordant, the connective tissue fiber of human tissue. etc., can also be dyed, thus obtaining an entirely homogeneous and beautiful image quality as in the double dyeing method. Also, dyeing allowance time is wide and the dying operation can be simplified so that up to mordant operation can be performed during immobilization process using, especially, an immobilizer composition for electron microscope.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a mordant used when observing a tissue of a living body such as a human using an electron microscope, and more specifically, it refers to a connective tissue fiber (a fiber in connective tissue) of a biological sample. The same shall apply hereinafter), and a mordant for electron microscope observation capable of finely staining with a suitable contrast and a method for staining a biological connective tissue sample for electron microscope observation using the same.

[0002]

2. Description of the Related Art When observing a tissue of a living body using an electron microscope and taking a photograph, staining is performed to give a contrast to a sample. This staining is usually performed by double-fixing an ultrathin section sample of living tissue with aldehydes such as glutaraldehyde and osmium tetroxide, and then embedding it in an epoxy resin to prepare an ultrathin section sample and quenching it with uranyl acetate. A method of double-staining with lead acid is used, but this method has a problem that connective tissue fibers are not clearly stained.

As a special electron staining method for solving this problem and staining connective tissue fibers for observing with an electron microscope, a tannic acid staining method by Kajikawa et al. [Kinichiro Kajikawa e
t.al .; J. Electron Microscopy 24 (4), 287-289 (197
5)] is widely known. This method is a method of improving the dyeability by immersing in a composition containing tannic acid as a main component before dyeing with uranyl acetate or lead citrate.

[0004]

However, in the tannic acid dyeing method, the contrast of the negative film photographed by an electron microscope is too strong, so that not only the connective tissue but other tissues are blurred, and the same connective tissue is used. Among the tissue fibers, there was an image defect that the fine structure portion was not visible because it was crushed by the shadow of the main structure.

In addition, it takes a long time for one night to prepare a mordant containing tannic acid as a main component, and the mordant prepared with difficulty is only stable for about one month at room temperature, and is easily discolored. The problem is that it must be prepared before use, which is a drawback in terms of operation and convenience. Further, if the processing time is not strictly observed, there are drawbacks in image quality such as a low degree of dyeing and an extremely blurred image, resulting in poor image quality.

Therefore, it has been desired to develop a mordant / staining method for a biological tissue sample which also stains connective tissue fibers and overcomes the operability, convenience and image defects of the above tannic acid staining method. It was

[0007]

[Means for Solving the Problem] When observing human tissues and the like using an electron microscope, the present inventors also stained fibrous tissues in connective tissues, and the image quality was doubled by uranyl acetate and lead citrate. As with the dyeing method, we conducted a thorough study to find a dyeing method that is uniform and beautiful throughout and has excellent operability.
As a result, they have found that if the tea leaf extract is used as a mordant and is used before double dyeing, all the above requirements are satisfied, and the present invention has been completed.

That is, an object of the present invention is to provide a mordant for electron microscope observation, which comprises a tea leaf extract. Another object of the present invention is to provide a method for staining a biological sample for electron microscope observation, which comprises using a tea leaf extract.

The mordant for electron microscope observation of the present invention (hereinafter,
Examples of the tea leaf extract used as an active ingredient of "mordanting agent for electron microscope" include non-fermented tea such as green tea, semi-fermented tea such as oolong tea, fermented tea such as black tea, and the like. It is preferably an extract of oolong tea.

The solvent used for the extraction of these tea leaves is water alone or a mixture of water and a lower alcohol such as methanol, ethanol or propanol or one or more polar solvents of lower ketones such as acetone or methyl ethyl ketone. Either is fine.

The ratio of tea leaf to solvent in the extraction is not particularly limited either, but it is about 2 to 1000 times by weight the solvent to 1 tea leaf, especially in terms of extraction operation and efficiency.
It is preferably about 0 to 100 times by weight. Furthermore,
The extraction temperature is conveniently in the range of room temperature to the boiling point of the solvent under normal pressure, and the extraction time varies depending on the extraction temperature.
It is preferably in the range of time to 1 week.

The tea leaf extract produced as described above may be used as it is as a mordant for electron microscopy.
It is desirable to use it as a powder formulation which is made into a dry powder by freeze-drying or the like, or a liquid formulation in which this dry powder is dissolved in a suitable solvent. The solvent used at this time may be any solvent used for extraction of tea leaves, but from the viewpoint of antiseptic property and storability, it is preferable that the solvent contains 10% by volume or more of a lower alcohol or a lower ketone.

The tea leaf extract prepared as described above may be further fractionated using a synthetic adsorbent column or the like, and a mordant for electron microscopy may be produced using the obtained fraction as an active ingredient. good. The preparation of the fraction of the tea leaf extract may be carried out, for example, according to the disclosure of JP-A-4-178320.

The mordanting agent for electron microscope of the present invention thus obtained is not only in a powder state but also in a state of being dissolved in the above-mentioned solvent, stable at room temperature for at least one year without being deteriorated, and a dyeing effect. No deterioration is observed. Further, a stable and good dyeing result can be obtained regardless of the mordant treatment time.

In order to prepare a biological sample for electron microscope observation (hereinafter referred to as "sample for electron microscope") using the above mordant for electron microscope, the following may be carried out. That is, first, a biological sample is fixed with aldehydes such as glutaraldehyde, p-formaldehyde, etc., using osmium tetroxide, etc., embedded in an epoxy resin, and cut into ultrathin sections.
Create a biological sample slice.

Then, the thin slice of the biological sample is immersed in the mordant for electron microscope of the present invention. The amount of the mordant for electron microscopy used in the mordant treatment of the biological sample slice is not particularly limited, but is preferably about 0.2 to 2.0% in terms of dry weight of the tea leaf extract.

To the mordant, p-nitrophenol or the like may be added to remove stains on the electron microscope image.
In addition, this mordant is very stable, even though the concentration of the tea leaf extract is relatively dilute, the dyeing effect does not deteriorate even after storage at room temperature for 2 months or more, and the growth of mold etc. is not observed. .

Further, the immersion time of the thin slice of the biological sample in the mordant for electron microscope may be about 5 to 15 minutes, and it is not necessary to control the immersion time very strictly.

Finally, a thin piece of a biological sample dipped in a mordant for electron microscopy can be double-stained according to a conventional method to obtain a sample for electron microscopy. This double staining can be performed by a generally used method, for example, a method using uranyl acetate and lead citrate.

As another method for preparing a sample for an electron microscope using the mordant for an electron microscope of the present invention, a mordant for an electron microscope is added to a fixing agent for electron microscope observation (hereinafter, referred to as "fixing agent for an electron microscope"). However, a method of simultaneously performing the fixing and the mordant treatment may be mentioned. In this method, for example, a fixative composition containing an aldehyde such as glutaraldehyde or p-formaldehyde and a tea leaf extract may be prepared.
It may be the same as the mordant for electron microscope. Further, the treatment time in this case may be the treatment time of the fixative.

[0021]

EFFECTS OF THE INVENTION By using the mordant for electron microscopy of the present invention, it becomes possible to stain connective tissue fibers such as human tissue which were not clearly stained only by the conventional double staining method using uranyl acetate and lead citrate, It is possible to obtain a homogeneous and beautiful image quality as in the double dyeing method.

Further, as compared with the tannic acid staining method (Kajikawa method) which is a conventional method for mordanting and staining connective tissue fibers, a fine structure of connective tissue fibers which could not be observed by the method can be observed. . Further, also in the preparation of the mordant for electron microscope, in the tannic acid dyeing method, it took time and effort to prepare the mordant for preparation before use, but in the case of the mordant for the electron microscope of the present invention, preparation before use was unnecessary. Therefore, the operability is remarkably improved as compared with the tannic acid dyeing method.

Furthermore, in the tannic acid staining method, it was necessary to strictly observe the staining time of 10 minutes, which sometimes used nerves. However, when the mordant for electron microscopy of the present invention was used, the allowable staining time was However, the dyeing operation has become simple, and it has become possible to mordant during the immobilization step, especially using the fixative composition for electron microscope.

[0024]

The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.

Example 1 Production Example of Tea Leaf Extract (1): 20 g of 100 g of oolong tea
The mixture was placed in a 00 ml Erlenmeyer flask, 1000 ml of hot water was added, and the mixture was boiled at 90 ° C for 15 minutes for extraction. This was filtered through Celite, the resulting filtrate was freeze-dried, and the extract 6.
5 g was obtained. With respect to black tea and pual tea, 16.4 g and 17.4 g of extracts were obtained in the same manner as above.

Example 2 Production Example (2) of tea leaf extract: 20 g of 100 g of oolong tea
The mixture was placed in a 00 ml Erlenmeyer flask, 1000 ml of 50% by volume ethanol was added, and the mixture was gently stirred every hour at room temperature for extraction for 3 hours. This was filtered through Celite, and the obtained filtrate was concentrated under reduced pressure to remove ethanol, water was added and freeze-dried to obtain 29.2 g of an extract. For black tea and puer tea, in the same manner as above, 30.4 g each,
31.3 g of extract was obtained.

Example 3 Mordant for Electron Microscope Observation (Liquid): 10 g of the oolong tea extract of Example 2 was dissolved in 100 ml of 40% by volume ethanol to give a mordant for electron microscope observation (liquid).

Example 4 Electron microscopic observation example: Human biopsy alveolar wall tissue was used as a biomaterial, which was double-fixed with 2.5% glutaraldehyde / 1% osmium tetroxide and then embedded in epoxy resin. Then, it was cut to prepare an ultrathin section sample. This section sample was immersed for 10 minutes in a mordant for electron microscope prepared by diluting the mordant prepared in Example 3 by 10 times with 50% by volume of ethanol.
Then, it was stained with a saturated aqueous solution of uranyl acetate for 10 minutes and with a lead citrate solution of Reynolds for 1 minute to prepare an electron microscope sample.

Uranyl acetate 2 was used as a control electron microscope sample.
Samples for electron microscopy prepared by ordinary double staining with 0 minute and 5 minutes with lead citrate and tannic acid staining by Kajikawa method were used, and then samples for double microscopy were used.

Each of the above-obtained samples was used for Hitachi H-7100.
It was observed with a scanning electron microscope (accelerating voltage 75 KV), and the dyeing properties of each collagen fiber and elastic fiber were compared and examined. The results are shown in FIGS.

As is clear from this result, in the case of double staining after staining with the oolong tea extract (FIG. 1), elastic fibers (fibers that appear dark in the upper center of FIG. 1) and collagen fibers (horizontal center of FIG. 1). Crested fibers) are clearly stained, and it is clear that the dyeing ability is superior to the case of double staining alone (FIG. 2).

Also, oolong tea extract and tannic acid have similar staining properties to connective tissue fibers, but at high magnification (2
In tannic acid staining, in particular, fine fibers found around the elastic fiber elastin were unclearly observed under tannic acid staining, whereas in the staining with oolong tea extract, These fine fibers were clearly observed (left part of the lower half of FIG. 3).

From the above, the electronic staining using the oolong tea extract is excellent in that the staining property in connective tissue fibers is similar to that of the conventional tannic acid staining, and the observation of the fine structure at high magnification is clear. It was shown to be a very useful method as a special electron staining method for observing connective tissue fibers.

Example 5 Effect of Oolong Tea Extract Concentration: Mordant (liquid) stock solution of Example 3 and this stock solution in 40 vol% ethanol 2,
Staining and observing human biopsy alveolar wall tissue was carried out in the same manner as in Example 4 using each of 5, 10, and 20-fold diluted solutions. As a result, a slight precipitation was observed when the stock solution and the 2-fold diluted solution were used. It was recognized that the results obtained using the 5, 10, and 20-fold diluted solutions were better.

Example 6 Effect of Mordanting Time: Human biopsy alveolar wall tissue was prepared in exactly the same manner as in Example 4 except that the mordanting time with the oolong tea extract was changed to 5, 10, 20, 30 and 40 minutes, respectively. When dyed and observed, a good quality image was obtained at any mordant time.

Example 7 Mordant with a composition obtained by adding a mordant for electron microscopy to a fixative:
2.5% diluted with 0.1M phosphate buffer (pH 7.4)
The supernatant obtained by mixing glutaraldehyde with the dry powder of the oolong tea extract obtained in Example 2 at a rate of 0.5% and filtering the mixture after it became saturated was used as a fixative composition for electron microscope. Then, the effect on mordant action and immobilization was investigated.

As the material, lung tissue of Wistar rat was used. Immediately after lung extraction, it was cut into 1 mm ³ squares and placed in the above-mentioned fixative composition for electron microscope. After fixing at 4 ° C. for 4 hours, it was post-fixed with 1% osmium tetroxide as a mold and embedded in an epoxy resin (Epok 812). As a control,
A tannic acid fixative prepared by mixing 2.5% glutaraldehyde with 1% tannic acid was used. Ultrathin sections were prepared, mounted on copper mesh, and then double-stained with uranium acetate for 5 minutes and lead citrate for 1 minute.
The staining state of lung tissue was evaluated with a Model 7100 electron microscope (accelerating voltage 75 KV). The results are shown in FIGS. 4 and 5.

From these results, the block dyeing effect of the fixative composition of the present invention containing the oolong tea extract (FIG. 4)
Is similar to that of the tannic acid-fixing solution used as a control (FIG. 5), and the collagen fibers (C) and elastic fibers (E) were clearly observed as having increased electron density. Furthermore, when the fixative composition of the present invention is used, the lamellar body (L) in the cytoplasm of epithelial cells of type II alveolar cells which is not clearly stained by the tannic acid fixative solution is also clearly stained. It was On the other hand, regarding the fixed state, the tannic acid fixative solution showed a slight conspicuous collapse of the intracellular organelles, whereas the oolong tea extract fixed solution showed good retention of the morphology of the intracellular organelles.

The reason for this is that the p of each fixative is
The difference in H, that is, the pH of the oolong tea fixative solution is 7.4 after preparation and the pH of the tannic acid fixative solution is 7.1. Generally, the optimum pH at which glutaraldehyde reacts with proteins is said to be higher than pH 7.4, and the reason why tannic acid fixative was improperly fixed compared to OTE fixative was due to the action of tannic acid. It is thought that this was due to the fact that it had become acidic.

From the above results, the glutaraldehyde fixative mixed with the oolong tea extract has a good fixing ability without the need to adjust the pH, and is also effective for connective tissue fibers such as collagen fibers and elastic fibers. Since it also has a strong mordant effect, it was revealed that it can be used as a highly useful fixative composition for electron microscope.

[Brief description of drawings]

FIG. 1 is an electron micrograph of a human biopsy alveolar wall tissue stained with a mordant for electron microscopy (oolong tea extract) of the present invention and then double-stained with uranyl acetate and lead citrate. Magnification × 9500; enlargement magnification × 5).

FIG. 2 is an electron micrograph of a human biopsy alveolar wall tissue double-stained with uranyl acetate and lead citrate (shooting magnification × 9500; enlargement magnification × 5).

FIG. 3 is an electron micrograph taken in the same manner as in FIG. 1 (shooting magnification × 30000; enlargement magnification × 5).

FIG. 4 is a photograph of a rat biopsy alveolar wall tissue stained with an electron microscopic fixative composition agent of the present invention (containing oolong tea extract and glutaraldehyde), and then double-stained with uranyl acetate and lead citrate. 3 is an electron micrograph (magnification × 9000; enlargement magnification × 5).

FIG. 5 is an electron micrograph of a rat biopsy alveolar wall tissue stained with a tannic acid-fixing solution and then double-stained with uranyl acetate and lead citrate (magnification × 900).
0; enlargement ratio × 5). that's all

Claims (5)

[Claims] 1. A mordant for electron microscopic observation of biological connective tissue fibers, which comprises a tea leaf extract. 2. The mordant for electron microscope observation according to claim 1, wherein the tea leaf extract is an oolong tea extract. 3. A method for staining a biological connective tissue sample for electron microscope observation, which comprises immersing the biological tissue sample in the mordant for electron microscope observation according to claim 1 prior to the double staining. 4. A biological connective tissue sample for electron microscope observation, which comprises fixing the biological tissue sample with the fixative for electron microscope observation containing the mordant for electron microscope observation according to claim 1, prior to double staining. Dyeing method. 5. A fixative composition for observing with an electron microscope, which comprises an aldehyde and a tea leaf extract.