Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

Our dissection method permits precise isolation of the ventricular neurogenic niche, and is therefore well-suited for studying the molecular microenvironment of this niche. The main advantage of this dissection method is that it's precise, efficient, and causes minimal tissue perturbation while still being compatible with mass spectrometry for proteome analysis. In this protocol, we extract the neurogenic niche of the ventricle from a mouse brain, but this method can also be applied to other species and in various states of health and disease.

The technique may require some training. especially with the scalpel cuts when exposing and extracting the ventricular neurogenic niche. After euthanizing an 8-to 10-week-old C57 black/6 male mouse, extract the brain by manual dissection and place it in a culture dish containing ice-cold dissection medium.

Using a scalpel, remove the olfactory bulb by make a straight coronal cut between the olfactory bulb and the interior pole of the cortex. Next, remove the anterior pole of the cortex by making a coronal cut, ensuring that the lateral ventricles are visible in the coronal plane. Then, using scissors, open both the lateral ventricles from the top, starting with the sagittal section from the cortical surface to the ventricular lumen.

Elongate this cut in a C-shaped manner following the ventricular flexion. Next, connect the caudal ends of the left and right sagittal incision, employing an additional coronal cut. Next, remove the cortex and corpus callosum covering the medial ventricular walls.

If the tissue is attached to the medial ventricular walls, make additional cuts or lift the cortex and corpus callosum with scissors to dislodge the tissue. Then, using forceps, remove the cortex and corpus callosum covering the lateral ventricles. Using forceps, carefully spread the ventricular walls and remove the choroid plexus.

Then, put the brain on a glass slide and place the slide on top of dry ice to freeze the brain with the ventricular walls in the open configuration. Before sectioning, ensure that the brain is attached to the cryostat attachment plate at the hind brain with an embedding medium for frozen tissues. Additionally, ensure that no embedding medium comes into contact with the fore brain, especially at the ventricles.

Then, cut 50-to 100-micrometer-thick coronal sections of the brain until the end of the lateral ventricle, and mount the sections on the glass slides. Place the glass slides on dry ice under a dissection microscope. Lift the slices from the dry ice for 15 to 30 seconds to achieve a brief, incomplete thawing, rendering the compact myelin of the striatum observable as dense white dots.

Then, using a pre-cooled scalpel, separate the subependymal zone from the adjacent striatum, and transfer it as a whole piece or sectioned into two to four parts into a microcentrifuge tube using the blunt edge of the cooled scalpel. Then do the same for the medial ventricular zone. Myelin-associated, glycoprotein-positive internal capsules of the striatum were identified in the wholemount samples, but rarely in the cryo-section-dissection samples via immunohistochemistry.

Striatal contamination in the wholemount samples was confirmed by enrichment of myelin proteins in the subependymal zone, compared to the somato-sensory cortex samples. In contrast, comparisons of these myelin marker proteins in the cryo-section-dissection samples showed no significant differences in the subependymal zone and somato-sensory cortex samples. When comparing mass spectrometry results between cryo-section-dissection and laser capture microdissection, laser capture microdissection yielded approximately half as many quantified proteins, although tissue collection time was approximately twice as long.

Principle component analysis reveals that there is larger variability among samples collected with laser capture microdissection, depicted as squares, than among those collected with cryo-section-dissection, depicted as circles. A 2D annotation enrichment test between cryo-section-dissection and laser capture microdissection for the subependymal and medial ependymal zones revealed similar enrichment in both methods and regions for proteins associated with the extracellular space. Cryo-section-dissection provides a more robust identification and quantification of neurogenesis in subependymal zone-associated extracellular matrix proteins.

In the case of tenascin-C, only cryo-section-dissection displayed enrichment in the subependymal zone compared to the medial ependymal zone. Make sure that the brain sections on the slides do not become completely thawed. Overall, it is good to practice the steps in the dissection procedure for a consistent result.

The dissection method can also be used with other protein analysis methods that can readily detect very low abundant proteins, such as growth factors and cytokines. This microdissection method allowed us to identify a new neurogenesis regulator, and we believe it will allow others to identify other regulators of neurogenesis in various contexts.