ACHE Stain (Copper Ferrocyanide Method)

Assay-kits

ACHE Stain (Copper Ferrocyanide Method)

Product code: ACMBS-39
Specifications: 8Tests, 20Tests (Box)
Intended use: Intended for histological acetylcholinesterase staining
Store temperature: 2℃～8℃
Expiry date: 12 Months

More Details

PrincipleMain componentsSample requirementTest procedureResult interpretationPrecautions

Principle

Acetylcholinesterase hydrolyses iodoacetyl thiocholine to release acetic acid and thiocholine. Sulfhydryl (-SH) in the thiocholine reduces potassium ferricyanide to potassium ferrocyanide, and potassium ferrocyanide combines with copper ions to form an insoluble cupric ferrocyanide precipitate in acetylcholinesterase parts.

Main components

Reagent composition	Main ingredient
1. Acetylthiocholine iodide powder	Acetylthiocholine iodide
2. Sodium acetate solution	Sodium acetate, glacial acetic acid
3. Sodium citrate solution	Sodium citrate
4. Copper sulfate solution	Copper sulfate
5. Potassium ferricyanide solution	Potassium ferricyanide

Sample requirement

Frozen sections: a thickness of 12～15μm, affixed to glass slides, dry with cold wind, or placed in a cold 10% solution of calcium formalin fixed for 15 min, washed with distilled water 3 times, 1 minute each.

Test procedure

A. Working solution preparation:

A working fluid volume of 12.1ml, can stain 4 to 6 slides at a time.

Reagents	Volume	Procedure
① Distill water (provide for oneself)	1.00mL	Pipette 1ml of ① into the bottle of ② and shake vigorously for 2 minutes to ensure that the powder in the bottle is fully dissolved and pour into the clean staining tank. Then ③, ④, ⑤, ⑥ in accordance with the amount of the above table added to the clean staining tank one by one, fully mixed, and put 37℃ pre-warmed, standby.
② Acetylthiocholine iodide powder	1 bottle
③ Sodium acetate solution	8.00mL
④ Sodium citrate solution	0.60mL
⑤ Copper sulfate solution	1.25mL
⑥ Potassium ferricyanide solution	1.25mL

B. Staining procedure:

1. Put the sections into the incubation solution, incubate at 37℃ for 1.5 hours, remove them when the sections are light brown, and rinse with distilled water.
2. Remove the sections and washed with running water slightly, then rinsed with distilled water.
3. Conventional dehydration and transparent, sealed with neutral resin.

Result interpretation

1. The active site of acetylcholinesterase is reddish brown or dark brown.
2. Rectal mucosa: According to the depth of staining, the relative number of positive nerves, and the thickness of positive nerves, the staining results were divided into negative, weak positive, positive and strong positive. Observed with 20-fold and 40-fold microscopy, none of the stained or lightly stained nerves was negative, and 3-10 positive nerves were weakly positive, positive nerves appeared fine mesh-like was positives, and positive nerves appeared dense mesh-like was strong positive.

Precautions

1. This product is suitable for frozen sections. When used, the chance of exposure at room temperature should be minimized. Otherwise, acetylcholinesterase is easily inactivated, resulting in false negatives.
2. This product can also be stained by the drop method, but it needs to be placed in a water bath and wet box for operation.
3. The reagent should be returned to room temperature before use, and the reagent should be shaken well, the container used to prepare the incubation solution must be clean.
4. The normal color of the incubation solution should be clear and transparent light green. If the incubation solution is cloudy, it cannot be used. It needs to be reconstituted.
5. The incubation solution needs to be used immediately. It is a one-time use and cannot be used repeatedly.
6. This reagent should be used by professionals and interpretation of the results. Read the instruction manual carefully before use, use it within the validity period, and do personal hygiene protection.
7. Dispose of waste according to the requirements of the hospital or environmental protection department after use.
8. The production date, production batch number and expiration date are shown in the outer packaging.

Reference

1. Ling Qi Bo. Practical pathology special staining and histochemistry technology [M]. Guangdong Higher Education Press.

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