The Stereology and 3D Volume Analyses in Nervous Tissue - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 34

without a random offset, it is easy to inadvertently choose more
completely stained sections and/or only sample at sites which are
not truly “statistically representative” within the ROI.

3.2. Increased
Reliability and Validity

Problems defi ning the ROI and counting parameters may infl uence
total cell counts and volume estimation. Reliability and validity of
cell counts or density measurements represent another bias present
with “2D” manual counting ( 7 ). This means that another counter
who repeats the same counting parameters and sampling would
arrive at a similar result. Stereological counting incorporates the
depth of a section, which provides additional information about
the volume of the selected area, whereas manual counting has limits
in these properties. In stereological counting, the optical disector
and fractionator, central components in stereology, have less bias in
assessing the number of the neurons within a section, but are subject
to sampling and estimation. Stereology protocols also require careful
attention to full antibody or counterstaining penetration throughout
the entire section thickness, something that is equally critical, but
often neglected when using other counting methods.

3.3. Controls
for Incomplete
Immunostaining

The depth of penetration of the antibody and counterstaining into
the tissue or lack thereof results in another counting bias. Non-
stereological counting, due to the limited attention to the z-axis
depth, will normally underestimate the number of cells when facing
incomplete staining through the section thickness. With stereology,
the section thickness and z -axis depth are sampled, which relies on
the ability to detect the immunostaining or counterstaining
throughout the entire section thickness, even though the actual
post-processed section thickness may be thinner than cut at the
microtome. Under such conditions, the total number of cells will
not be underestimated with proper staining. Several factors dis-
cussed below infl uence antibody penetration.
Antibody penetration may be infl uenced by tissue fi xation, cutting
thickness, use of detergents such as Triton X100 to increase pene-
tration, and the incubation time and method (e.g., free fl oating vs.
slide mounted). For all staining methods, penetration issues can be
a confounding factor for using the optical disector in stereology
( 8 ). One aspect not mentioned in this study ( 8 ) is that one needs
to have optimized their immunostaining protocols and system,
while taking the limitations of immunostaining into consideration.

The method chosen to cut tissue may infl uence several factors that
are important for stereology. The issues to be concerned about are
antibody penetration and z-axis shrinkage. Z-axis shrinkage alters
the distribution of cells depending on the fi xation and cutting
method used ( 9 ). According to the study, vibratome, paraffi n, and
glycolmethacrylate plastic sections have higher cell densities at the

3.4. Infl uences of
Tissue Processing