JPH0711520B2 - How to make a permanent sample of cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a permanent specimen of cells, tissues and the like (herein, these are referred to as cells).

(Prior Art) With the development of medicine, immunohistochemical staining methods have been widely used. Conventionally, antibodies labeled with peroxidase (hereinafter referred to as POX) are often used, but it is necessary to inactivate the POX present in the sample prior to staining, and the oxygen generated in this operation is necessary for cells and specimens. May collapse. In particular, when applied to a smear sample of bone marrow or peripheral blood or a stamp sample of lymph node, cell damage is remarkable.

In the POX labeling method, a benzidine compound that produces a stable dye is used as a substrate, but since the produced dye is brown, it is difficult to distinguish it from an in vivo dye such as melanin. And no suitable substrate has yet been found to produce stable dyes.

As a method of using a labeling enzyme other than POX, a method of using alkaline phosphatase (hereinafter referred to as AP) is known. The advantage of the method using AP is that (1) it is not necessary to inactivate the enzyme existing in the sample in a normal paraffin-embedded section. (2) The substrate used is not carcinogenic. (3)
By combining a naphthol-based substrate and a coupler of a diazonium salt, a dye having excellent color contrast can be obtained.
(4) The reaction on the alkaline side is less likely to cause dissociation of the antigen-antibody reaction. On the other hand, a drawback is that the produced dye is soluble in an organic solvent and cannot be encapsulated in anhydrous form, and is encapsulated in glycerin jelly or the like, which causes fading in several months.

(Problems to be Solved by the Invention) The present invention solves the drawbacks of the conventional method of labeling cells with AP to prepare a specimen, enables water-insoluble resin encapsulation, has less fading, and localizes the dye. It is intended to provide a method of producing a permanent specimen having excellent properties.

(Means for Solving the Problem) In the present invention, cells labeled with AP are treated with AP substrate at a pH of 8.2 to 8.5.
The reaction product produced by reacting the reaction product with the diazotized product of basic fuchsin to deposit an organic solvent-insoluble red dye, which is then encapsulated with a water-insoluble resin encapsulant. It is a method for producing a permanent sample of cells, which is characterized in that

The cells that can be stained by the method of the present invention can be any cells as long as they can be labeled with AP, and specifically, lymph muscle, spleen, bone marrow, liver, intestinal tract, nerves. , Kidneys, tissues such as endocrine glands and blood,
Examples include free cells such as ascites fluid, cerebrospinal fluid, and alveolar lavage fluid.

In addition, examples of the form of the sample used in the method of the present invention include paraffin-embedded sections, frozen sections, smear samples, and cytocentrifuge samples.

As a method for labeling cells with AP, any method known per se can be applied. For example, a direct method in which AP is directly bound to an antigen in a cell to label the antigen, a primary antibody is reacted with an antigen in the cell, and a biotinylated secondary antibody is further reacted to the secondary antibody. There is an indirect method in which AP is bound and labeled.

As a method for binding AP to an antibody, for example, a method by chemical bonding such as glutaraldehyde or periodate oxidation method,
Any known method such as a method of binding via an intermediary such as an AP anti-AP method can be adopted. These enzyme labeling methods include, for example, the enzyme antibody method (Interdisciplinary Planning Publishing, March 1985).
Issued on 25th).

The primary antibodies that can be used in the present invention include rabbit anti-human specific antibodies (anti-human immunoglobulin, anti-human lysozyme, anti-human carcinoembryonic protein, anti-human keratin,
Anti-human leukocyte common antigen), mouse anti-human specific monoclonal antibody, etc., and secondary antibodies include goat anti-rabbit immunoglobulin, rabbit anti-mouse immunoglobulin, etc. You can

Further, as a substrate for AP that can be used in the present invention, naphthol AS-MX phosphate, naphthol AB-BI phosphate,
Examples include α-naphthylphosphoric acid, naphthol AS-GR and salts thereof.

Furthermore, examples of the basic fuchsin used as a coupler for reacting with an enzymatic reaction product include compounds represented by the following formulas. These compounds are
Used after diazotization.

Following the above color development reaction, it is also possible to perform nuclear staining in blue with hematoxylin. According to this method, cells are stained red and nuclei are stained blue, so that the color contrast becomes clear and the cells can be easily identified. It should be noted that other dyeing methods can be appropriately adopted as necessary.

By performing the above enzyme reaction in a buffer solution with a pH range of 8.2 to 8.5, the dissociation of the antigen-antibody reaction can be suppressed, the diffusion of the formed dye can be prevented, and the red color and the organic Since a solvent-insoluble reaction product can be produced, the stained cells can be encapsulated in a water-insoluble resin.

The cells after the color reaction are treated with isopropyl alcohol or the like, and the paraffin-embedded tissue is further treated with
Perform xylol treatment and directly for other samples,
Encapsulate with a water-insoluble resin encapsulant such as Avi Will Becker's Diatex or Merck's Enteran New.

(Action) The present invention allows AP labeled cells to act on the AP substrate and react the resulting reaction product with the basic fuchsin diazotized product to produce an organic solvent insoluble red dye. This is a method of depositing this on cells to stain the cells, dehydrating them, and then encapsulating with a water-insoluble resin encapsulating agent to prepare a permanent specimen. The dye generated here has excellent localization to cells, does not diffuse the dye after being deposited on cells, and enables water-insoluble resin encapsulation, that is, anhydrous encapsulation, and is a permanent dye that does not fade for a long period of time. Allows creation of specimens. Further, when blue counternuclear staining with hematoxylin is performed, the blue color of the nucleus forms a clear contrast with the red color of the tissue, which facilitates the identification of cells. Furthermore, it became possible to perform double staining with POX labeling.

(Example 1) After fixing lymph muscle with 20% formalin, a permanent specimen was prepared using a tissue embedded in paraffin.

First, on a slide glass pretreated with a 0.1% neoprene toluene solution, a slice of the above-embedded tissue was sliced to a thickness of 4 μm, extended, dipped in xylol and deparaffinized, and then with a phosphate buffer solution. The sample was prepared by washing.

Then, smear 5% normal goat serum, react at room temperature for 15 minutes, wash once with phosphate buffer, and smear rabbit anti-human immunoglobulin as primary antibody at room temperature.
The mixture was reacted for 60 minutes and washed again with a phosphate buffer. Then, as a biotinylated secondary antibody, biotinylated goat anti-rabbit immunoglobulin manufactured by Vector Co., Ltd. was applied, reacted at room temperature for 30 minutes, washed with a phosphate buffer, and further, as avidin labeled with alkaline phosphatase. Was applied with Bio-Yeda alkaline phosphatase-labeled avidin, reacted at room temperature for 30 minutes, and then washed with a phosphate buffer to obtain AP-labeled cells.

On the other hand, as a substrate, naphthol AS-BI phosphate 10 mg 0.2
It was dissolved in ml of dimethylformamide. Further, 4 g of Fuchsin NB as a coupling solution was dissolved in 100 ml of 2N hydrochloric acid,
A 0.1 ml filtrate was collected by filtration, 4 g of sodium nitrite was dissolved in 100 ml of distilled water, and 0.1 ml of Fuchsin NB solution and sodium nitrite were collected and mixed to give a yellow-brown transparent liquid. Obtained. This clear liquid was added with 9.6 mg of L-levamisole 0.2
Dissolve in 40 ml of M Tris-HCl buffer (pH 8.5) and add 0.2m to it.
l of substrate solution was added and stirred, and then filtered.

In the obtained filtrate, the AP-labeled cells obtained above were immersed and reacted at room temperature for 15 minutes, and after washing with a phosphate buffer,
The cells were fixed with 20% formalin solution and kept at room temperature for 10 minutes, and then treated with hematoxylin solution for 2 minutes at room temperature to stain nuclei blue.

Then, treat with isopropyl alcohol,
It was dehydrated with xylol and resin-sealed with Diatex to obtain a permanent specimen.

The permanent specimen thus obtained was stained red and showed excellent localization, and because the nucleus was stained blue, the contrast became clear and the identification of cells became easier. Since it was encapsulated in anhydrous and insoluble in xylol, no fading was observed for a long time.

In addition, for samples of embedded tissues of spleen, bone marrow, liver and intestinal tract, permanent samples were prepared in the same manner as above,
Results similar to those of the above-mentioned permanent lymph muscle specimen were obtained.

(Example 2) Regarding embedded tissue after fixing 20% formalin of bone marrow, as a substrate in the procedure of Example 1, naphthol AS-BI sodium phosphate 10 mg was directly added to 9.6 mg of L-levamisole 0.2.
A permanent sample was prepared in the same manner as in Example 1 except that the solution was dissolved in 40 ml of M Tris-HCl buffer (pH 8.5).

The obtained specimen was subjected to vivid counterstaining in the same manner as in Example 1, and a permanent specimen without fading for a long period of time with easy tissue identification could be obtained.

Example 3 A sample was prepared by fixing a smear of peripheral blood with anhydrous acetone, holding it for 5 minutes, drying, and washing with a phosphate buffer.

Then, the treatment was promoted according to the procedure of to in Example 1 to prepare AP-labeled cells, which were double-stained, treated with isopropyl alcohol, and immediately mounted with Diatex.

The obtained specimen did not show any cell damage, was subjected to bright counterstaining as in Example 1, and a permanent specimen without fading for a long period of time and with easy cell identification could be obtained.

In addition, a permanent sample was prepared by the same procedure as above for the smear of bone marrow, the stamp of lymph muscle, the cytocentrifuge of ascites, and the frozen sections of lymph muscle, spleen, bone marrow, liver and intestine. Similar results were obtained with a permanent sample of peripheral blood.

(Advantages of the Invention) The present invention, by adopting the above-mentioned constitution, can stain with a dye insoluble in an organic solvent, so that a water-insoluble resin can be encapsulated and an excellent permanent specimen that does not fade for a long time can be obtained. It is now possible to provide. Further, the above dye enables staining with excellent localization to cells, and
Since this dye has a bright red color, it is clearly distinguished from the color of the tissue, the contrast becomes vivid when the nucleus is stained blue, and the cells can be identified more easily.

Claims (3)

[Claims] 1. Formed by reacting alkaline phosphatase-labeled cells with an alkaline phosphatase substrate in a buffer solution having a pH range of 8.2 to 8.5, and reacting the generated reaction product with a basic fuchsin diazotized product. A method for producing a permanent specimen of cells, which comprises depositing an organic solvent-insoluble red pigment and encapsulating the pigment with a water-insoluble resin encapsulating agent. 2. A method comprising reacting a primary antibody with a light source in a cell, further reacting with a biotinylated secondary antibody, and binding alkaline phosphatase to the secondary antibody for labeling. The method for producing a permanent specimen of cells according to claim 1. 3. The method for producing a permanent sample of cells according to claim 1 or 2, wherein blue nuclear staining with hematoxylin is performed before encapsulating with a water-insoluble resin encapsulating agent.