In the 1970s, Kristensopn and Olsson reported that horseradish peroxidase (HRP) can be taken up by nerve terminals, transported retrogradely via axoplasm to the neuronal soma, and the outline of neurons can be visualized by histochemical methods. Based on this finding, they developed the HRP neuron tracing technique, namely the HRP method. DAB, short for 3,3'-Diaminobenzidine Tetrahydrochloride, is a common substrate for horseradish peroxidase. Catalyzed by HRP, DAB produces a brown precipitate that is insoluble in water and ethanol, presenting a distinct brown color after chromogenic reaction and being observable under a microscope.
Neuronal HRP Tracing Chromogenic Solution (DAB Method) works on the principle that after animals are anesthetized and injected with HRP, free or complexed HRP reacts with an oxidant to form a complex. This complex then oxidizes the hydrogen-donating DAB chromogenic agent, resulting in a brown product that is clearly visible under a microscope. This reagent is intended for research purposes only and not for clinical diagnosis or any other applications.
N1508662
| Component | 50T | Storage |
| N1508662A | DAB Assay Buffer | 2×500 mL | RT. Store in the dark. |
| N1508662B | DAB Chromogenic Solution
| 30 mL | -20℃. Store in the dark. |
| N1508662C | DAB Enhancer
| 2×1 mL | 2-8℃. Store in the dark. |
| N1508662D | DAB Wash Buffer (20×) | 100 mL | RT. |
Operating Procedures (For Reference Only)
(1) Preparatory Work
1. Animal Anesthesia: 3.5% sodium pentobarbital is commonly used as the anesthetic. The anesthetic dose for rats is 0.25-0.35 mL per 100 g body weight.
2. HRP Administration: Methods include pressure injection, electrophoresis, and topical application/injection for the peripheral nervous system.
3. Determine Animal Survival Period: Set the appropriate survival time post-HRP administration according to experimental requirements.
4. Animal Perfusion: After anesthesia, perform intracardiac perfusion fixation via cannulation of the ascending aorta through the left ventricle: First, perfuse rapidly with physiological saline or phosphate-buffered saline (PBS). Next, perfuse with 4% paraformaldehyde fixative, starting at a fast rate then slowing down, with the total duration controlled at 30-40 minutes. Finally, perfuse with 10% sucrose phosphate buffer (pH 7.4).
5. Tissue Harvesting and Sectioning: Immerse harvested tissues in 20% sucrose phosphate buffer. Cut tissues into sections with a thickness of 40 μm and store the sections in sucrose phosphate buffer for later use.
(2) Chromogenic Reaction
1. Prepare DAB Incubation Solution: Mix an appropriate volume of DAB Assay Buffer and DAB Chromogenic Solution at a ratio of 39:1. This solution must be prepared fresh and used immediately; it is not suitable for storage.
2. Prepare DAB Working Chromogenic Solution: Mix the freshly prepared DAB Incubation Solution with DAB Enhancer at a ratio of 2000-8000:1. The optimal ratio should be determined empirically according to specific experimental conditions. This working solution must be prepared fresh and used immediately; storage is not recommended.
3. Prepare 1× DAB Wash Buffer: Dilute the 20× DAB Wash Buffer with distilled water at a ratio of 1:19. The 1× working solution can be stored at room temperature and is valid for 6 months.
4. Wash Sections: Rinse the tissue sections with distilled water 3 times, 2 minutes each time.
5. Incubate with DAB Incubation Solution: Immerse sections in 10 mL of pre-warmed (20℃) DAB Incubation Solution, incubate for 20 minutes in the dark with continuous gentle agitation.
6. Incubate with DAB Working Chromogenic Solution: Transfer sections to 10 mL of pre-warmed (20℃) DAB Working Chromogenic Solution, incubate for 20 minutes in the dark with continuous gentle agitation.
7. Rinse Sections: Rinse sections with approximately 10 mL of 1× DAB Wash Buffer 2-3 times, 5 minutes each time.
8. Mount Sections: Mount sections onto glass slides pre-coated with chrome alum gelatin, then air-dry at room temperature.
9. Dehydration and Clearing: Perform the following steps sequentially:
① Distilled water: 10 seconds.
② 70% ethanol: 10 seconds.
③ 95% ethanol: 10 seconds.
④ 100% ethanol: 2 times, 10 seconds each time.
⑤ Xylene or clearing agent: 2 times, 2-5 minutes each time.
10. Coverslipping and Observation: Seal sections with neutral balsam and observe the brown reaction product under a microscope.
Precautions
1. High background staining or precipitate formation indicates an overly intense DAB substrate reaction; adjust the reaction conditions or reagent ratios accordingly.
2. All labware must be thoroughly clean to avoid contamination with oxidants or reducing agents, which may cause non-specific reactions.
3. Avoid repeated freeze-thaw cycles of the DAB Chromogenic Solution, as this will reduce its chromogenic efficiency.
4. Store the DAB Enhancer in a tightly sealed container; improper sealing will lead to decreased chromogenic activity.
5. For your safety and health, wear a lab coat and disposable gloves during all operations.
6. Use the reagents as soon as possible after opening to prevent compromising subsequent experimental results.
