For the frontal lobe of this particular subject a total of 760 sections were systematically collected with 76 stained for cresyl violet.In this example we were interested in the frontal lobe and have defined it as the region from the tip of the frontal pole (anterior) to the central sulcus (posterior) and the lateral sulcus (ventral) and excluded the insula. The entire reference space must be well defined from anterior to posterior and dorsal to ventral.Section Sampling Fraction (non-computer based).
The next sections of this protocol show how to determine the ssf, asf, tsf and ∑ Q. Where ssf is the section sampling fraction, asf is the area sampling fraction, tsf if the thickness sampling fraction (where the measured thickness of the tissue is divided by the dissector height), and ∑ Q- is the total number of objects of interest counted within the dissector.
All other series were banked in antigen preserve as part of our long-term research plans. Series 2 was split so that half of the sections were stained with gold chloride for myelin revelation and the other half stained for acetylcholine esterase (for delineation of certain subcortical areas). 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. Briefly, tissue should be well perfused with paraformaldehyde, glutaraldehyde, or formalin.Part 1: Pre-processing of tissue should be done according to Burke et al. In addition to contrast and comparison of results from both the BioQuant and Stereologer systems, this study provides a detailed protocol for the Stereologer system. This study documents a biological application of computerized stereology to estimate the total neuronal population in the frontal cortex of the vervet monkey brain ( Chlorocebus aethiops sabeus), with assistance from two commercially available stereology programs, BioQuant Life Sciences and Stereologer (Figure 1). 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.