MARK WEST STEREOLOGY SERIES
One complete series was stained with cresyl violet (series #1). For our purposes the series were set at 1/10 sections throughout the cortex, sectioned at 50µm. The reference space was defined to include cortical tissue from the central sulcus to the frontal pole of the left hemisphere. In this example we focused on the frontal lobe of the vervet brain.Part 2: Systematic Sampling of tissue, according to Burke et al.
The brain should be stereotaxically blocked, removed from the skull, weighed, volume determined, cryoprotected, and frozen 5. Tissue should be well perfused with paraformaldeyhde, gluteraldehyde, or formalin. This is followed by a 4% paraformaldehyde solution in PBS for 5 min (~1 liter). In the present study the subject was deeply sedated with ketamine hydrochloride (10 mg/kg, i.m.), euthanized with an overdose of sodium pentobarbital (25 mg/kg, i.v.) and perfused transcardially with 0.1 M PBS until completely exsanguinated. This can be achieved through standard transcardial perfusion typically used to harvest other organs.
Among the advantages of these stereological approaches over previous methods is the avoidance of all known sources of systematic (non-random) error arising from faulty assumptions and non-verifiable models. The key components of the approach are unbiased (systematic-random) sampling of anatomically defined structures (reference spaces), combined with quantification of cell numbers and size, fiber and capillary lengths, surface areas, regional volumes and spatial distributions of biological objects within the reference space 4. Unbiased stereology, the method accepted as state-of-the-art for quantification of biological objects in tissue sections 2, generates reliable structural data for biological features in the mammalian brain 3. The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain.