Histochemical Imaging of Cortical Modules in Flattened Brain Sections

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.