Histochemical Imaging of Cortical Modules in Flattened Brain Sections

Samenvatting

Source: Lauer, S. M., et al. Visualization of Cortical Modules in Flattened Mammalian Cortices. J. Vis. Exp. (2018)

This video demonstrates a method to visualize the cortical modules in flattened brain sections using histochemical staining. Pre-fixed sections are treated with a straining reagent that highlights active neural clusters, forming dark, high-contrast regions. After mounting and dehydration, cortical modules appear under a bright-field microscope as dark, patchy patterns.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Visualization of Cortical Modules Using Cytochrome Oxidase Staining

NOTE: Different staining protocols have been developed for histochemical detection of cytochrome oxidase activity. This protocol uses nickel-ammonium sulfate (NiAS), resulting in a higher contrast and better-defined modules in stained cortical areas.

1. Prepare the cytochrome oxidase staining solution. For 10 mL of solution, add 10 mL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (0.1 M, pH 7.4), 400 mg sucrose, 12.5 mg NiAS, 2 mg cytochrome C, and 6 mg diaminobenzidine (DAB).
   CAUTION: DAB and NiAS are carcinogenic.
   NOTE: Add DAB just prior to incubation of sections.
2. Wash the sections in HEPES buffer (0.1 M, pH 7.4) on a shaker for 15 min.
3. Incubate the sections in the staining solution at room temperature on a shaker.
   NOTE: Observe the speed of the staining. If there is no visible reaction, change to incubation at 37 °C. Depending on the amount of fixation, staining can be observed after 10 min or in several hours.
4. Stop the reaction by adding 4% paraformaldehyde (PFA); this prevents unwanted re-dying and an increase of background signal.
5. Wash the sections three times using the HEPES buffer for 10 min.
6. Mount and dry the sections on glass slides.
7. Dehydrate the sections with an increasing alcohol row:
   1. Wash the slides in 60% ethanol for 1 min. Wash the slides in 80% ethanol for 1 min. Wash the slides in 96% ethanol for 2 min. Wash the slides in 100% ethanol for 3 min. Wash the slides in isopropanol for 5 min. Wash the slides in xylene for 5 min.
8. Immediately add a quick hardening-mounting medium and add a coverslip. Critical: Do not use water-based mounting mediums, as NiAS will be washed out and degrade the staining.
9. Keep the sections at 4 °C for long-term storage.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Cytochrome oxidase staining
Cytochrome c from equine heart	Sigma-Aldrich	C2506
3,3'Diaminobenzidine tetrahydrochloride hydrate	Sigma-Aldrich	D5637
HEPES	Carl Roth	9105.4
Mounting or freezing media
Eukitt (histochemistry)	Sigma-Aldrich	3989
Alcohol dehydration
Ethanol 100%	Carl Roth	9065.3
Ethanol 96%	Carl Roth	P075.3
2-Propanol	Carl Roth	6752.4
Xylene substitute	Fluka	78475
Devices/tools
Dumont 55 Forceps - Inox	Fine Science Tools	11255-20
Dumont 5 Forceps - Inox Biology Tip	Fine Science Tools	11252-30
Dumont 5SF Forceps - Inox Super Fine Tip	Fine Science Tools	11252-00