This article details a staining protocol for visualizing cortical neurons using diaminobenzidine (DAB) and nickel-ammonium sulfate. The method enhances contrast in brain sections, allowing for the identification of active neural clusters involved in sensory processing.
Start with pre-fixed tangential sections of the flattened brain cortical sections. Wash to remove fixative traces.
Stain the sections with a solution of diaminobenzidine or DAB and nickel-ammonium sulfate.
DAB diffuses into cortical neurons and reacts with cytochrome oxidase in mitochondria, forming a dark precipitate. Nickel-ammonium sulfate darkens the precipitate, enhancing contrast.
Add paraformaldehyde to stop the staining reaction, then rinse.
Mount the sections on a slide and dehydrate them with ethanol washes.
Rinse with isopropanol and xylene to clear the section and improve light transmission.
Apply a mounting medium and put a coverslip on it.
When imaged under a bright-field microscope, within the cortical neurons, the precipitates absorb and scatter light, making them appear dark.
In contrast, unstained, cleared areas transmit light and appear bright.
This contrast highlights cortical modules as dark, patchy structures against a lighter background, representing active neural clusters involved in processing sensory information.
Wash the section tissue in HEPES buffer for 15 minutes with gentle agitation. During the wash, prepare fresh cytochrome oxidase staining solution, and add the DAB component just before using it. Now, incubate the sections in the freshly made staining solution at room temperature with shaker. And keep watch. Depending on the amount of fixation, staining could be observable in 10 minutes, or it may take several hours.
If the reaction is going too slow, increase the temperature to 37 degrees Celsius. To stop the reaction, transfer the sections to a bath of 4% PFA, and let them incubate for 15 minutes with gentle agitation. Then, wash the sections three times with HEPES buffer for 10 minutes per wash.
Now, mount and dry the sections on glass slides, and dehydrate them using progressively stronger ethanol baths. Then, wash the slides in isopropanol for 5 minutes, followed by xylene for five minutes. After the xylene bath, immediately add a quick hardening non-water based mounting medium. Water-based mediums will degrade the stain. After adding a coverslip, store the slides at 4 degrees Celsius until imaged.
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