ATPase Staining Kit for Muscle Fibers (Calcium Activation Method) 

Adenosine Triphosphatase (ATPase) is a hydrolase enzyme that catalyzes the hydrolysis of ATP to release energy. Depending on the activators, inhibitors, and localization of the enzyme, ATPase can be classified into membrane-bound ATPase, myosin ATPase, mitochondrial ATPase, etc. Myosin ATPase has an optimal pH of 9.2-9.4 and is activated by calcium ions, while magnesium ions inhibit its activity. The energy released by ATP hydrolysis is used for muscle contraction. This enzyme is primarily located in skeletal muscle and is commonly used to differentiate between different types of muscle fibers.

Type I fibers (Red muscle) are slow-twitch fibers with slower contraction speeds but are capable of sustained activity over a long period. They have higher ATPase activity and exhibit darker staining. Type II fibers (White muscle) are fast-twitch fibers with faster contraction speeds but shorter durations of activity. They have lower ATPase activity and show lighter staining.

The reaction for ATP hydrolysis catalyzed by ATPase is as follows:

A-P-P-P + H₂O → A-P-P + H₃PO₄ + Energy

MCE ATPase Staining Kit for Muscle Fibers (Calcium Activation Method) is based on the hydrolysis of ATP by ATPase, which generates ADP and inorganic phosphate. Phosphate combines with calcium ions to form colorless calcium phosphate precipitates. Calcium phosphate is then treated with cobalt chloride to form cobalt phosphate, which, after treatment with sulfide solution, results in the formation of brown-black cobalt sulfide precipitates at the enzyme activity sites.

This kit contains acidic and basic incubation solutions. When the sample is pre-incubated with the basic solution, only white muscle fibers show a positive reaction, which is highly specific. When pre-incubated with the acidic solution, red muscle fibers will stain. Therefore, the calcium activation method for ATPase staining is primarily used to differentiate between red (slow-twitch) and white (fast-twitch) muscle fibers.

Acidic Incubation Solution, Basic Incubation Solution, ATPase Incubation Solution A, ATPase Incubation Solution B, ATPase Sulfide Solution: 4°C, 6 months. Protected from light.

ATPase Cobalt Solution: RT, 6 months.

Reagent Preparation

1. Samples: Frozen sections of skeletal muscle and other tissues

2. Equipment: Water bath or incubator

3. Reagents: Distilled water, graded ethanol, neutral resin, xylene, etc.

Procedure

1. Tissue Section Preparation: Obtain fresh tissue and prepare frozen sections using low-temperature cryosectioning (liquid nitrogen freezing provides better results). The recommended thickness of the sections is 6 μm. Cut the tissue into continuous sections and place them on glass slides, labeling them as Sample A and Sample B. The sections do not require fixation.

2. Preparation of ATPase Incubation Solution: Mix equal volumes of ATPase Incubation Solution A and ATPase Incubation Solution B, adjusting the pH to 9.3-9.5.

Note: It is recommended to prepare the ATPase incubation solution fresh for each use.

3. Preparation of ATPase Sulfide Working Solution: Dilute the ATPase Sulfide Solution 50-100 times with distilled water.

Note: It is recommended to prepare the ATPase sulfide working solution fresh for each use.

4. Pre-incubation of Solutions: Pre-incubate the acidic incubation solution, basic incubation solution, and ATPase incubation solution at 37°C for 10 min.

5. Tissue Section Treatment

5.1 Sample A Treatment: Immerse Sample A in the acidic incubation solution and incubate at 37°C for 5 min, followed by a 5-second rinse in water. Then, transfer it to the basic incubation solution and incubate at 37°C for 30 s, followed by another 5 s rinse in water.

5.2 Sample B Treatment: Immerse Sample B in the basic incubation solution and incubate at 37°C for 15 min, followed by a 5 s rinse in water.

6. ATPase Incubation: Immerse both samples in the ATPase incubation solution and incubate at 37°C for 30-60 min, followed by a 5 s rinse in water.

7. ATPase Cobalt Solution Incubation: Immerse the sections in the ATPase cobalt solution and incubate for 3 min, followed by a 5 s rinse in water.

8. Sulfide Working Solution Treatment: Immerse the sections in the sulfide working solution and incubate for 30-60 s, followed by a 30 s rinse in water.

9. Routine Dehydration, Clearing, and Mounting: Perform routine dehydration and clearing procedures, followed by mounting with neutral resin or mounting medium.

Staining Interpretation

The ATPase-stained sections typically appear black or dark brown.

Muscle Fiber Types and Staining Results

1. Type I (Red muscle): Deep staining with acidic incubation solution, light staining with basic incubation solution.

2. Type IIa (White muscle): Light staining with acidic incubation solution, deep staining with basic incubation solution.

3. Type IIb (White muscle): Moderate staining with acidic incubation solution, deep staining with basic incubation solution.

1. The activity of ATPase is significantly affected after fixation, so the tissue sections should not be fixed during the staining process.

2. After mixing, the pH of the ATPase incubation solution is close to 9.4, which is suitable for enzyme staining. If conditions permit, it is recommended to check and adjust the pH to the appropriate range before use. If the pH deviates significantly or if precipitates form, the staining may fail, so the solution should be freshly prepared each time.

3. ATPase sulfide solution is corrosive and has an irritating odor. It should be handled with caution and used in a fume hood. After each use, ensure that the bottle is tightly closed to prevent evaporation and loss of activity.

4. Once prepared, the ATPase sulfide working solution will degrade and lose its effectiveness over time. Therefore, it is recommended to prepare only small quantities as needed.

5. When performing staining on frozen sections, minimize the exposure time of the sections at room temperature to ensure optimal staining results.

6. The staining outcome is closely related to the pH of the acidic incubation solution, basic incubation solution, and ATPase incubation solution. Efforts should be made to minimize factors that could alter the pH, such as frequently opening the containers or leaving the sections at room temperature for extended periods.

7. Reagents should be used as soon as possible after opening to avoid potential effects on experimental results.

8. This product is for R&D use only, not for drug, household, or other uses.

9. For your safety and health, please wear a lab coat and disposable gloves to operate.