JPH01237452A - Composition of coloring liquid and manufacture thereof and manufacture of preparation used in said composition - Google Patents

Abstract

PURPOSE:To prevent fading due to light from a coloring material, to improve a shelf life and to implement high coloring density, by dissolving Stains-all into the mixed solvent of nitroparaffin and low alcohol. CONSTITUTION:A material 1-ethyl-2-(3-(1-ethylnaphto(1,2-d)thiazolin-2-ylidene)-2- methylpropenyl)naphto(1,2-d)thiazolium bromide shown in the formula) (Strains-all in short) is suspended in nitroparaffin. Then low alcohol is added and dissolved. When the Stains-all is dissolved in this way, a sample can be dyed at a sufficient density. Fading with light is negligible, and the shelf life is excellent. The sufficient density can be obtained in the composition of the coloring liquid having the composition rate such as this as the coloring liquid for preparation up to about 2wt.% in general.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a novel dyeing liquid composition, a method for producing the same, and a method for producing preparations using the same.

Conventional techniques and their problems A preparation involves fixing a specimen on a glass slide in order to observe its fine structure using an optical microscope or the like. For example, preparations for microscopy such as pathological tissue specimens are manufactured using conventional methods. That is, the tissue to be examined is fixed in formalin → dehydrated with alcohol and then xylene → embedded in paraffin → a tissue section is prepared with a microtome → the section is placed on a slide glass and stretched → dehydrated with xylene and then alcohol. paraffin →
It is manufactured through the following process: dyeing it in various ways → dehydrating it with alcohol → immersing it in xylene to clear it → coating it with resin if necessary and then encapsulating it.

Among the above steps, staining is conventionally performed using hemadoxylin-eosin, RAS (periodic acid Schiff), toluidine blue,
This is done using a staining solution such as Alcian blue, but there are some tissues, such as sialomucin and melanin, that can be hardly or completely stained with the conventional staining solution such as 2nd class, and it is difficult to improve the staining properties of the staining solution. This is considered to be an important issue in histopathological examinations, etc.

On the other hand, 1-ethyl-2-(3-(1
-ethylnaphtho(1,2-d)thiazolin-2-ylidene)-2-methylpropenyl)-naphtho(1,2-d)
Thiazolium bromide (common name: StainS-8ll, hereinafter referred to as StainS-ol) is known (J, Ho1. Biol, 4).
1,139 (1969), JHi stochem,
Cytochem, 22.767 (1974)
).

In other words, Stainzuol of the above formula has excellent stainability for nucleic acids, proteins, complex proteins, etc., and can stain proteins red, phosphoproteins blue, nucleic acids purple, and mucoproteins and mucopolysaccharides in various colors. It is known to stain (J, His
tochem, Cytochem, , 22.76
7 (1974)), and is expected to be used as a dye for tissues such as pathological tissues.

However, in the past, stain-zo-all was not prepared.
Attempts have been made to use borumamide, ethylene glycol, etc. for staining the specimens listed above (J, Biol, Chem.

10, 5985 (1985), etc.), and its solubility is at most 0.5% by weight, which has the disadvantage that it is not possible to dye with sufficient concentration. Furthermore, when these solvents are used, there is a drawback that the color of the dyed product is significantly faded by light, and for example, when exposed to sunlight, the color completely fades in only a few minutes, resulting in extremely poor storage stability. Furthermore, the stained specimen had the disadvantage that not only was the staining concentration low, but when xylene was used in the clearing process as usual, stain-sur-ol would be eluted and the staining concentration would become even thinner and almost disappear.

As mentioned above, due to the various drawbacks mentioned above, it is currently impossible to use Stain Zuol as a staining solution for preparations.

Means for Solving the Problems In view of the above-mentioned current situation, the inventors of the present invention have conducted extensive research in order to eliminate the various drawbacks of Stain-Z-All as a staining solution for preparations. As a result, it was found that when stinzuol is dissolved in a specific mixed solvent, it can be made into a staining solution with a sufficient concentration as a staining solution for preparations, that the staining solution can be suitably produced by a specific method, and that the staining solution can be used to stain specimens. It can be dyed to a sufficient concentration, and surprisingly, there is almost no fading due to light and it has good storage stability.If a specific mixed solvent is used in place of xylene in the clearing process, there is almost no elution of stain-zool. The present invention was completed based on these findings.

That is, the present invention provides a staining solution composition prepared by dissolving stenzul in a mixed solvent of nitroparaffin and lower alcohol, which involves suspending stainzul in 21-21~lobarafuin, and then adding and dissolving the lower alcohol. A method for producing the dyeing liquid composition described above, and a preparation characterized in that in producing the preparation, the dyeing liquid composition is used for dyeing, and a mixed solvent of p-cymene and cyclohexane is used for transparency. Relating to the manufacturing method.

The dyeing liquid composition of the present invention is made by dissolving stainzol in a mixed solvent of nitroparaffin and lower alcohol.

Here, examples of the nitroparaffin include nitromethane, nitroethane, 1-nitropropane, 2-nitropropane, 1-nitrobutane, and 2-nitrobutane, and at least one of these is used. Among these, nitromethane and nitroethane are preferred from the viewpoint of high solubility in steinzool as a mixed solvent with a lower alcohol. In addition, examples of lower alcohols include methanol, ethanol, n-butanol,
Examples include isobutanol, n-propatool, isopropatool, etc., and at least one of these is used. Among these, it is preferable to use methanol or ethanol.

The ratio of the nitroparaffins and lower alcohols to be used is usually about 3 to 6:4, preferably about 4:6 to 5:5, in terms of volume ratio of the former to the latter.

Further, the concentration of stain-all in the dyeing solution composition of the present invention can be generally about 2% by weight, which is sufficient as a staining solution for preparations. Steinzol is hardly soluble in nitroparaffin or lower alcohol alone, but it is soluble in a mixed solvent of these, and this fact is true. This was discovered for the first time by the inventor.

The stain solution composition of the present invention can be suitably prepared by suspending stainsuol in nitroparaffin, and then adding and dissolving a lower alcohol. This is presumed to be because, when pretreated with nitroparaffin, stainsuol, which was originally slightly soluble in lower alcohols, becomes easily soluble due to the solvation effect of nitroparaffins and lower alcohols. More specifically, first, a predetermined amount of steinsuol is suspended in nitroparaffin, and although there is no particular restriction, it is mixed and stirred for usually about 10 minutes to 24 hours, preferably about 15 minutes to 2 hours, and then a lower alcohol is added. In addition, although there is no particular restriction, the dye solution composition of the present invention having a desired concentration of up to about 2% by weight can be prepared by stirring for usually about 10 minutes to 24 hours, preferably about 15 minutes to 3 hours. can.

The staining solution composition of the present invention thus obtained has a sufficient concentration as a staining solution for preparations, so that it can stain specimens with a sufficient concentration, and surprisingly, there is almost no fading due to light. For example, as shown in the Examples below, preparations obtained using the composition of the present invention have sufficient storage stability. This is presumed to be because nitroparaffin has some kind of photobleaching prevention effect.

Furthermore, the dyeing solution composition of the present invention has excellent storage stability as such. For example, as shown in Example 2 below, when 2% by weight Sting-All is stored in an amber bottle, there is no change in concentration even if it is left for 5 months.

When manufacturing a preparation using the dyeing solution composition of the present invention, it can be carried out in the same manner as the conventional manufacturing process described above, except that the composition is used as the dyeing solution.

Therefore, since the staining solution composition of the present invention can be used to stain the specimen on the preparation to a sufficient density, it is also possible to use xylene for clearing, although the staining density will be lower.

However, when a specimen is stained using the stain composition of the present invention, it is preferable to use a mixed solvent of p-cymene and cyclohexane in place of xylene in the clearing step. This prevents the dyeing density from becoming too thin and allows for sufficient penetration.

This mixed solvent has never been used as a clarifying agent and was discovered for the first time by the present inventor.

The mixing ratio of p-cymene and cyclohexane is usually about 3 to 7:3, preferably about 4:6 to 5:5 in consideration of volatility.

Furthermore, when a specimen is stained using the stain composition of the present invention, any conventionally known mounting medium such as balsam can be used in the mounting process of the preparation, but the present inventor has succeeded in developing it first. It is preferable to use the mounting medium filed as Application No. 61-240666.

That is, the general formula [In the formula, R1 does not represent a hydrogen atom or a methyl group.

R2 and R3 are the same or different and represent a lower alkyl group, or one is a lower alkyl group and the other is -CH2C
Represents an R' group or a -CH2CH2C-R' group. Here, R4 represents a methyl group or a phenyl group. ] When using a photocurable mounting medium for preparations containing a 1,3-dioxolane derivative as an active ingredient, simply place the mounting medium on the specimen on a slide glass and irradiate it with light under anaerobic conditions.
Preparations with excellent light resistance and storage stability can be easily and quickly produced.

Preferred specific examples of the 1,3-dioxolane derivatives of the above general formula (I) include 2-methyl-2-acetonyl-1,3-dioxolan-4-yl)methyl acrylate, <2-methyl-2- Acetonyl-1,3-dioxolan-4-yl)methyl methacrylate, (2-methyl-2-(β-acetylethyl)-1,3-dioxolan-4-yl)methyl acrylate, (2-medy-2-(β) -acetylethyl)-1,3-dioxolane-
4-yl)methyl methacrylate, (2-methyl-2-
benzoylmethyl-1,3-dioxolan-4-yl)
Examples include methyl acrylate, (2-methyl-2-benzoylmethyl-1,3-dioxolan-4-yl)methyl methacrylate, and the like. In addition, in the mounting medium for preparations, for example, 1-hydroxycyclohexylphenyl ketone, pentyl dimethyl ketal,
A photopolymerization initiator such as an aromatic ketone compound such as benzoin isobutyl ether, benzoin ethyl ether, 2,2-jethoxyacetonoenone, benzoin isopropyl ether, or benzophenone is added to 100 parts by weight of the above 1,3-dioxolane derivative. It is usually desirable to add about 0.005 to 2 parts by weight.

Specimens for preparing preparations using the stain composition of the present invention are not particularly limited, but include, for example, human pathological or normal tissue specimens, cytology specimens, animal and plant pathological or normal tissue specimens, microbial specimens, etc. can be mentioned.

Effects of the Invention According to the present invention, for the first time, there has been provided a dyeing solution composition using Stainzuol as a dye, which has conventionally been practically impractical as a dyeing solution for preparations, a method for producing the same, and a suitable method for producing preparations using the same. A remarkable effect is produced.

By using the staining solution composition of the present invention, it is possible to obtain dyeing properties in preparations that have not been previously available. For example, in the case of pathological tissue specimens from animals such as humans, sialomucin is stained blue when observed under an optical microscope. Furthermore, melanin granules are divided into those that are stained purple, those that are stained blue-black, and those that are not stained. Also, mast cells are stained purple. Also, under a fluorescence microscope, amyloid emits coral-colored fluorescence.

Example Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Dyeing liquid compositions of the present invention were prepared using the following formulations. For preparation, a predetermined amount of stinzool is first mixed with a predetermined amount of nitroparaffin at room temperature, stirred for 15 to 30 minutes, and then a predetermined amount of lower alcohol is added and stirred for 15 to 15 minutes.
This was done by stirring for a period of time to dissolve.

0 staining solution istainzuol 2g nitromethane 50ml 1 ethanol
50ml 1O staining liquid Stainzuol 2g Nitroethane 50ml Ethanol
50ml0 staining solution hastainzol 2g nitromethane 40ml ethanol
60ml0 Staining solution 1g Nitromethane 50ml Ethanol
50ml0 Staining solution Hostein Zuol 0.50 Nitromethane 50ml Ethanol
501 0 staining solution 2g 2-nitropropane 50ml ethanol
5 (IIO staining staining staining 2q 2-nitropropane 50ml methanol
50ml Example 2 When the stain solution A obtained in Example 1 was stored tightly in an amber bottle, there was no change in concentration at all even after 5 months. still,
Concentration changes were determined by absorbance at 570 nm.

Example 3 Slide glass (thickness 1.2mm, width 76mm.

The appendix tissue of the mouse was processed on a 25 mm vertical specimen according to the usual procedure for preparing pathological tissue specimens, stained by immersion in the staining solution A prepared in Example 1 for 10 minutes at room temperature, dehydrated, and then treated with p-cymene and cyclohexane. (2-methyl-
A mounting medium containing 0.5 parts by weight of 1-hydroxycyclohexyl phenyl ketone mixed with 100 parts by weight of 2-acetonyl-1,3-dioxolan-4-yl) methyl acrylate was added dropwise, and a cover glass (. ,15X24x
24 mm) were stacked one on top of the other to avoid air bubbles. After lightly pressing the top of the cover glass with a cotton swab, light irradiation was performed to obtain a preparation of the tissue specimen according to the present invention.

For comparison, a comparative tissue sample preparation was obtained in the same manner as above, except that it was stained with hematoxylin and eosin and cleared with xylene.

Using an optical microscope, each prepared specimen was subjected to weak magnification (1 to 99 times), moderate magnification (100 to 200 times),
Observations were made under high magnification (201 to 400 times) and when using an oil immersion lens (1000 to 2000 times). FIG. 1 is a micrograph of a preparation of a tissue specimen according to the present invention (
200 times). Moreover, FIG. 2 is an example of a micrograph (200x magnification) of a preparation of a tissue specimen for comparison. As is clear from comparing FIG. 1 and FIG. 2, sialomucin (spherical portion) is stained (actual color is blue) with the composition of the present invention, but hematoxylin-
Sialomucin is not stained with eosin staining and appears white.

Color photographs of Figures 1 and 2 are attached as reference drawings.

Furthermore, when a storage stability test was carried out on the preparation obtained according to the present invention according to the accelerated stability test conditions described in 1 Pharmaceutical Manufacturing Guidelines 1986j (published by Japan Official Standards Association, published by Yakuji Jihosha), no changes were observed. It was extremely stable.

Example 4 A pathological skin tissue of Lille melanosis was used as a pathological specimen, the staining solution prepared in Example 1 was used as a staining solution, and an equal volume mixture of p-cymene and cyflohexane was used as a clearing solution. A preparation of a tissue specimen according to the present invention was obtained in the same manner as in Example 3 except that

For comparison, a comparative tissue sample preparation was obtained in the same manner as above, except that it was stained with hematoxylin and eosin and cleared with xylene.

Each of the obtained preparations was observed using an optical microscope in the same manner as in Example 3. FIG. 3 is a micrograph (200x magnification) of a preparation of a tissue specimen according to the present invention.
This is an example. Moreover, FIG. 4 is an example of a micrograph (200x magnification) of a preparation of a tissue specimen for comparison. As is clear from comparing Figures 3 and 4, melanin is darkly stained with the composition of the present invention (actual color is blue-black), but with hematoxylin-eosin staining, melanin is almost completely stained. Not stained.

Color photographs of Figures 3 and 4 are attached as reference drawings.

Further, when the storage stability test for the preparation obtained according to the present invention was conducted in the same manner as in Example 3, no change was observed and it was found to be extremely stable.

Example 5 A pigmented nevus (dermal nevus) skin pathological tissue was used as a pathological tissue specimen, the staining solution prepared in Example 1 was used as the 4 staining solution, and p-cymene, cyclohexane, etc. were used as the clearing solution. A preparation of a tissue specimen according to the present invention was obtained in the same manner as in Example 3 except that a volumetric mixture was used.

For comparison, a comparative tissue sample preparation was obtained in the same manner as above, except that it was stained with hematoxylin and eosin and cleared with xylene.

Each of the obtained preparations was observed using an optical microscope in the same manner as in Example 3. FIG. 5 is a micrograph (200x magnification) of a preparation of a tissue specimen according to the present invention.
This is an example. Moreover, FIG. 6 is an example of a micrograph (200x magnification) of a preparation of a tissue specimen for comparison. As is clear from a comparison of Figures 5 and 6, melanin is darkly stained with the composition of the present invention (actual color is blue-black), but melanin is almost stained with hematoxylin-eosin staining. It has not been.

In addition, color photographs of Figures 5 and 6 are attached as reference drawings of Figures 5 and 6.

Further, when the storage stability test for the preparation obtained according to the present invention was conducted in the same manner as in Example 3, no change was observed and it was found to be extremely stable.

[Brief explanation of the drawing]

FIG. 1, FIG. 3, and FIG. 5 show that in Examples 3 to 5,
It is a micrograph of a preparation obtained using the staining solution composition of the present invention. FIG. 2, FIG. 4, and FIG. 6 show that in Examples 3 to 5,
It is a micrograph of preparation = 22- obtained using a comparative staining solution. (Above) Fig. 1 Fig. 3 Fig. 2 Fig. 4 Fig. 5 Fig. 6] Indication of the case Patent Application No. 64906 of 1988 2 Name of the invention Dyeing liquid composition, method for producing the same, and use thereof Preparation method for preparations 3 Relationship with the case of the person making the amendment Patent applicant Sanpo Chemical Research Institute Co., Ltd. 4 Agent June 28, 1988 6 Contents of the amendment subject to amendment 1 Brief explanation section on the Chinese side of the specification Correct the description as follows. [Figures 1, 3, and 5 are micrographs showing the morphology of organisms in preparations obtained using the stain compositions of the present invention in Examples 3 to 5. FIG. 2, FIG. 4, and FIG. 6 show that in Examples 3 to 5,
It is a micrograph showing the morphology of organisms in preparations obtained using a comparative staining solution. ” (hereinafter “2”)

Claims (3)

[Claims] (1) Add 1-ethyl-2-[3-(1-ethylnaphtho[1,
A staining liquid composition prepared by dissolving 2-d]thiazolin-2-ylidene)-2-methylpropenyl]-naphtho[1,2-d]thiazolium bromide. (2) 1-ethyl-2-[3-(1-ethylnaphtho[1
,2-d]thiazolin-2-ylidene)-2-methylpropenyl]-naphtho[1,2-d]thiazolium bromide is suspended in nitroparaffin, and then a lower alcohol is added to dissolve it. A method for producing a dyeing solution composition according to claim 1. (3) Claim 1 for dyeing when manufacturing preparations
A method for producing a preparation, characterized by using the staining liquid composition described above and using a mixed solvent of p-cymene and cyclohexane for transparency.