JP2006187274A - Method for rapidly and histochemically staining acetylcholine esterase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for rapidly and histochemically staining acetylcholine esterase. <P>SOLUTION: The method for rapidly and histochemically staining the acetylcholine esterase comprises adding a solution A containing iodized acetylcholine, a citric acid salt and copper sulfate, and a solution B containing potassium ferricyanide to a frozen tissue fragment having 3-6 μm thickness, incubating the resultant fragment at 10-30°C for 4-6 min, adding a diaminobenzidine salt and hydrogen peroxide to the incubated product, further carrying out the incubation at 10-30°C for 1-2 min, and washing the product with water. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a rapid histochemical staining method of acetylcholinesterase useful for staining nerve cells and nerve fibers.

  Acetylcholine is an important neurotransmitter, and the concentration of acetylcholine in the brain is reduced in Alzheimer's disease. Further, in intestinal dysfunction (including constipation), an abnormal increase in acetylcholinesterase is observed. Acetylcholinesterase is an enzyme that hydrolyzes acetylcholine into choline and acetic acid. Staining and identifying acetylcholinesterase activity is extremely useful as an indicator of whether or not nerve cells and nerve fibers are normally present and functioning. is important. As one of the means for staining the acetylcholinesterase activity, the histochemical staining method is widely used in the areas of clinicopathological tissue diagnosis and animal experiments in the cranial nerve region and intestinal nerve region.

  A conventional acetylcholinesterase histochemical staining method is prepared immediately before use by preparing a solution A containing acetylthiocholine iodide, citrate, copper sulfate and isooctamethylpyrophosphorylamide and a solution B containing potassium ferricyanide. A liquid and B liquid are mixed, and a tissue section having a thickness of about 10 μm is added thereto and incubated at 37 ° C. for 60 minutes. Subsequently, osmium tetroxide was added, incubated at room temperature for 10 minutes, and then washed with water (Non-patent Document 1).

In this method, the entire dyeing process takes a long time of 70 minutes, and it is necessary to prepare liquid A and liquid B immediately before use, and since powder may be used as a material to be used, there is a risk of toxicity due to inhalation or the like. was there. Therefore, the present inventors mixed the tissue section with the above solutions A and B, then incubated at 37 ° C. for 5 minutes, added diaminobenzidine salt and hydrogen peroxide, incubated at room temperature for 1 to 2 minutes, and then washed with water. And found that histochemical staining of acetylcholinesterase can be performed in a short time (Non-patent Document 2).
Arch. Pathol. Lab. Med. 1978; 102: 244-247 Arch. Pathol. Lab. Med. 1994; 118: 1127-1129

Although the above method has the advantage that all steps of staining can be completed in a short time, the staining is insufficient and there is a background, so it is not yet satisfactory in terms of nerve cell specific staining. It was.
Accordingly, an object of the present invention is to provide an acetylcholinesterase histochemical staining method capable of specifically staining nerve cells in a short time and with a simple operation.

  Therefore, the present inventor made the thickness of the frozen tissue section, which was 10 μm in the past, to be 3 to 6 μm, and added the sections to the A solution and B solution which were made into kits, and then at 10-30 ° C. instead of 37 ° C. By incubating for 4 to 6 minutes and thinning the tissue section, the permeability of the staining solution is increased and a nerve cell-specific stained image can be obtained. Also, by reducing the solution penetration time, the background (single And the present invention has been completed.

That is, the present invention adds a solution A containing acetylthiocholine iodide, citrate, and copper sulfate, and a solution B containing potassium ferricyanide to a frozen tissue section having a thickness of 3 to 6 μm. A rapid acetylcholinesterase histochemical staining method characterized by incubating at 4 ° C. for 4-6 minutes, then adding diaminobenzidine salt and hydrogen peroxide, incubating at 10-30 ° C. for 1-2 minutes, and then washing with water It is.
In the present invention, a solution A containing acetylthiocholine iodide, citrate and copper sulfate, and a solution B containing potassium ferricyanide are added to a frozen tissue section having a thickness of 3 to 6 μm at 10 to 30 ° C. And 4 to 6 minutes, followed by adding a diaminobenzidine salt and hydrogen peroxide, incubating at 10 to 30 ° C. for 1 to 2 minutes, and then washing with water.

  According to the method of the present invention, since all the steps can be performed near room temperature, a device such as a thermostat is not required, all steps are completed in a short time of 5 to 8 minutes, and nerve cells are specifically stained. Therefore, hematoxylin-eosin staining was used to determine the normal part (the part where nerve cells are recognized) during the radical operation of Hirschsprung's disease. However, since frozen sections are used, the nerve cells should be determined. Although it has been reported that postoperative complications often occur due to a diagnosis error, it is possible to identify neurons reliably during surgery by using this staining method. became. In the future, effective use for not only the above-mentioned diseases but also for diseases for which nerve cells must be identified is considered. Moreover, it is thought that by preparing the liquid A and the liquid B in advance and making them into a kit, the stability of the solution is enhanced, and the success rate of dyeing is considered to be significantly increased, and the operability is further improved.

  The tissue used in the present invention may be a tissue in a nerve region of an animal including a human, for example, a tissue in a cranial nerve region or an enteric nerve region. The collected tissue is frozen and cut into 3 to 6 μm thick sections. Conventionally, a section of about 10 μm was used, but by using a section of 3 to 6 μm, the background (monocyte staining and the like) disappeared, and the nerve cells became clearer. The thickness of the section is more preferably 4 μm.

  Tissue sections are incubated for 4-6 minutes at 10-30 ° C. in addition to solution A containing acetylthiocholine iodide, citrate and copper sulfate, and solution B containing potassium ferricyanide. In this reaction, hydrogen produced by the decomposition of acetylthiocholine iodide by acetylcholinesterase present in the tissue reacts with potassium ferricyanide, copper sulfate, and citrate to produce copper ferrocyanide, which is generated by nerve cells. It is a reaction that deposits on the acetylcholinesterase site. Here, the solution A containing acetylthiocholine iodide, citrate and copper sulfate, and the solution B containing potassium ferricyanide are solutions containing acetylthiocholine iodide, citrate and copper sulfate immediately before the test. It is desirable to prepare by mixing A and solution B containing potassium ferricyanide.

  The concentration of acetylthiocholine iodide in the solution A is preferably 1-2 mg / mL, particularly 1.12 mg / mL. As the citrate, sodium citrate is preferable, and the citrate concentration in the solution A is preferably 10 to 15 mM, particularly preferably 11.2 mM. The concentration of copper sulfate in the liquid A is preferably 5 to 7 mM, particularly preferably 6.6 mM. Furthermore, it is preferable to add 100-200 mM of acetate buffer (pH 6.0), especially 145 mM to A liquid.

  The B liquid preferably contains 1 to 2 mM, particularly 1.1 mM potassium ferricyanide.

  The liquid A and liquid B are preferably prepared in advance so as to be prepared as a kit so that they can be prepared immediately before use.

  In the present invention, frozen tissue sections are added to the A + B solution and incubated at 10-30 ° C. for 4-6 minutes. Since it is only necessary to incubate at a room temperature of 10 to 30 ° C., there is no need to use a thermostat or the like. The incubation time is preferably 5 minutes.

  After the above incubation, diaminobenzidine salt and hydrogen peroxide are added and incubated at 10-30 ° C. for 1-2 minutes. This reaction is a step of coloring the copper ferrocyanide deposited on the acetylcholinesterase moiety by the above reaction, and the color development is completed in a short time of 1 to 2 minutes by using a diaminobenzidine salt and hydrogen peroxide together. .

  As the diaminobenzidine salt, diaminobenzidine tetrahydrochloride is preferable, and the final concentration used is preferably 1 to 2 mM, particularly preferably 1.5 mM. The hydrogen peroxide concentration is preferably 0.01 to 0.02% by mass, particularly 0.015% by mass. Further, it is preferable to add 50 mM Tris-HCl (pH 7.6) to a liquid containing a diaminobenzidine salt and hydrogen peroxide.

  Incubation is preferably for 1 minute at room temperature.

  After the incubation, the tissue may be washed with water and then observed with a microscope. As the microscope, it is preferable to use an optical microscope that is usually used everywhere.

  The obtained stained image is specific to nerve cells, has a small background compared to the conventional staining method, and is extremely clear. Moreover, since all the processes are completed in about 6 minutes near room temperature, diagnosis can be performed during the operation.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples at all.

Example 1
Ten patients with Hirschsprung's Disease (HD) were subjected to radical pull-through surgery, and the colon tissue of the excised specimen was collected. A healthy subject's large intestine tissue was used as a control. The tissue was frozen with liquid nitrogen. First, the prepared liquid A contains acetylthiocholine iodide at a concentration of 1.12 mg / mL, sodium citrate at 11.2 mM, and copper sulfate at 6.6 mM, and acetate buffer (pH 6.0) at 145 mM is added. Has been. On the other hand, the B liquid contains 1.1 mM potassium ferricyanide. Liquid A and liquid B were mixed, and a tissue section having a thickness of 4 μm was added thereto and incubated at room temperature (25 ° C.) for 5 minutes.
The tissue section was washed with water and then added to a solution containing 50% Tris-HCl (pH 7.6) containing 0.015% by mass of hydrogen peroxide (final concentration: 1.5 mM) in diaminobenzidine tetrahydrochloride. Incubated for 1 minute at room temperature (25 ° C.). The tissue section was then washed with water.

The obtained section was observed with a microscope. FIG. 1 shows an acetylcholinesterase-stained image of a Hirschsprung disease patient. According to the method of the present invention, it can be seen that nerve fibers are selectively and clearly stained in mucosal villi and submucosal tissues by a simple operation for a total of 6 minutes. Also, unlike the conventional quick method, there is no background at all.
On the other hand, when a tissue section having a thickness of 10 μm was used and the incubation was performed at 37 ° C. in a mixed solution of solution A and solution B, the background was strong (especially in the mucous villi). Many monocytes are stained.), Staining selectivity of nerve cells was not sufficient. In addition, nerve fibers in the villi characteristic of Hirschsprung's disease patients are not stained at all (FIG. 2).

It is a figure which shows the colon tissue acetylcholinesterase histochemical staining image of a Hirschsprung's disease patient. It is a figure which shows the colon tissue acetylcholinesterase histochemical dyeing | staining image of the Hirschsprung's disease patient using the thing of thickness 10 micrometers as a tissue section.

Claims (4)

  A solution A containing acetylthiocholine iodide, citrate and copper sulfate, and a solution B containing potassium ferricyanide were added to a frozen tissue section having a thickness of 3 to 6 μm and incubated at 10 to 30 ° C. for 4 to 6 minutes. And then adding a diaminobenzidine salt and hydrogen peroxide, incubating at 10 to 30 ° C. for 1 to 2 minutes, and then washing with water, followed by a rapid acetylcholinesterase histochemical staining method.   A solution A containing acetylthiocholine iodide, citrate and copper sulfate, and a solution B containing potassium ferricyanide were mixed with solution A containing acetylthiocholine iodide, citrate and copper sulfate immediately before the test. The rapid acetylcholinesterase histochemical staining method according to claim 1, which is prepared by mixing with solution B containing potassium ferricyanide.   A solution A containing acetylthiocholine iodide, citrate and copper sulfate, and a solution B containing potassium ferricyanide were added to a frozen tissue section having a thickness of 3 to 6 μm and incubated at 10 to 30 ° C. for 4 to 6 minutes. And then adding a diaminobenzidine salt and hydrogen peroxide, incubating at 10 to 30 ° C. for 1 to 2 minutes, and then washing with water.   A solution A containing acetylthiocholine iodide, citrate and copper sulfate, and a solution B containing potassium ferricyanide were mixed with solution A containing acetylthiocholine iodide, citrate and copper sulfate immediately before the test. 4. The method for staining nerve cells according to claim 3, wherein the method is prepared by mixing with solution B containing potassium ferricyanide.