In the 1970s, Kristensopn and Olsson reported that horseradish peroxidase (HRP) can be taken up by nerve terminals, transported retrogradely via axoplasm to neuronal somata, and the outlines of neurons can be visualized by histochemical methods. Based on this finding, the HRP neuron tracing technique, namely the HRP method, was developed. 3,3',5,5'-Tetramethylbenzidine (TMB) is an excellent chromogenic reagent for enzyme-linked immunosorbent assays (ELISAs). It is soluble in various organic solvents and double-distilled water, presenting as a stable colorless solution. When mixed with an appropriate amount of urea peroxide or hydrogen peroxide and buffer solution, TMB reacts with peroxidase to produce a distinct blue product that is highly observable. Catalyzed by horseradish peroxidase, TMB generates a blue precipitate that is insoluble in water and ethanol, displaying a blue color after chromogenic reaction for microscopic observation.
Neural HRP Tracing Chromogenic Solution (TMB Method) is designed for the following experimental process: after animals are anesthetized and injected with HRP, free or complexed HRP reacts with an oxidant to form a complex. This complex oxidizes the TMB chromogenic reagent, resulting in a blue color that is clearly visible under a microscope. This detection method is more sensitive than the DAB method.This chromogenic solution is intended for research use only and not for clinical diagnosis or other purposes.
N1508680
| Component | 50T | Storage |
| N1508680A | TMB Assay Buffer | 500 mL | 2-8℃. Store in the dark. |
| N1508680B | TMB Chromogenic Solution
| 15 mL | 2-8℃. Store in the dark. |
| N1508680C | TMB Enhancer
| 2×1 mL | 2-8℃. Store in the dark. |
| N1508680D | TMB Wash Buffer (20×) | 100 mL | RT. |
Procedure (For Reference Only)
(1) Pre-Experiment Preparation
1. Animal Anesthesia: Anesthetics such as 3.5% sodium pentobarbital, diazepam, or 10% chloral hydrate are commonly used. For rats, the anesthetic dose of sodium pentobarbital is 0.25-0.35 mL per 100 g of body weight.
2. HRP Administration: Methods include pressure injection, electrophoresis, and topical application in the peripheral nervous system.
3. Determine Animal Survival Time.
4. Animal Perfusion: After anesthesia, perform intracardiac perfusion fixation via cannulation of the ascending aorta from the left ventricle. First, perfuse rapidly with normal saline or PBS.
Subsequently, perfuse with 4% paraformaldehyde fixative at a rapid rate initially and then slow down, with the total perfusion time controlled at 30-40 min. Finally, perfuse with 10% sucrose phosphate buffer (pH 7.2-7.4).
5. Tissue Sampling: Place the harvested tissues in 20% sucrose phosphate buffer. Cut sections at a thickness of 40 μm and store them in sucrose phosphate buffer for later use.
(2) Chromogenic Reaction
1. Prepare TMB Incubation Solution: Mix an appropriate volume of TMB Assay Buffer and TMB Chromogenic Solution at a ratio of 39:1. The TMB incubation solution must be prepared immediately before use and should not be stored.
2. Prepare 1× TMB Wash Buffer: Dilute the 20× TMB Wash Buffer with distilled water at a ratio of 1:19. The 1× working solution can be stored at room temperature for up to 6 months.
3. Wash the sections with distilled water 3 times, 2 min each time.
4. Immerse the sections in 10 mL of pre-warmed TMB incubation solution (20℃). Incubate for 20 min in the dark with continuous shaking.
5. Prepare TMB Chromogenic Working Solution: Remove the sections and directly add TMB Enhancer to the used TMB incubation solution at a ratio of 2000-8000:1 (the optimal ratio should be optimized according to specific experimental conditions). The working solution must be prepared immediately before use and should not be stored.
6. Re-immerse the sections in the freshly prepared TMB chromogenic working solution. Incubate at 20℃ for 20 min in the dark with continuous shaking.
7. Rinse: Rinse the sections with approximately 10 mL of 1× TMB Wash Buffer 2-3 times, 5 min each time.
8. Mount the sections: Use slides pre-coated with chrome alum gelatin. Air-dry the slides at room temperature.
9. Dehydration and Clearing Steps:
① Distilled water: 10 s.
② 70% ethanol: 10 s.
③ 95% ethanol: 10 s.
④ 100% ethanol: 2 times, 10 s each time.
⑤ Xylene or deparaffinization clearing solution: 2 times, 2-5 min each time.
10. Mount the sections with neutral mounting medium and observe the blue reaction product under a microscope.
Precautions
1. High background staining or precipitation indicates an excessively intense TMB substrate reaction.
2. The formation of blue flocculent particles in the TMB chromogenic working solution after preparation is a normal phenomenon. Continuous shaking is required during use to prevent the particles from depositing on the sections and interfering with subsequent observation.
3. All utensils must be thoroughly cleaned to avoid contamination with oxidants or reducing agents, which may cause non-specific reactions.
4. Avoid repeated freeze-thaw cycles of the TMB Chromogenic Solution. The TMB Enhancer should be stored in a sealed container to prevent the reduction of chromogenic efficiency.
5. The Wash Buffer contains volatile components; tightly cap the bottle after use to prevent volatilization and loss of efficacy.
6. For your safety and health, wear a lab coat and disposable gloves during operation.
7. Use the reagents promptly after opening to avoid compromising subsequent experimental results.
