The Stereology and 3D Volume Analyses in Nervous Tissue - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 35

top and bottom of sections due to shrinkage, but have a central
valley region with fewer cells. Celloidin sections, tissue embedded
in a nitrocellulose solution and cut on a sliding microtome, had
minimal z-axis distortion. Cryosections, on the other hand, bulged
more at the center with less cell density at the top and bottom ( 9 ).
Further studies are needed to confi rm these results when replicated
by other investigators.
Cutting tissue inadvertently causes another bias present in
manual counting. Parts of cells at the top or bottom of the section
may be cut off during cutting. This phenomenon, known as “lost
caps,” causes underestimation of the population ( 7 ). Stereology
corrects for “lost caps” by requesting users to specify a guard zone
at the top and bottom of the section that is not counted (see
Methods below). Appropriate guard zone sizes are highly variable
and should be determined empirically on a case-by-case basis during
a pilot study.
4. Preparing
for Stereology
The following two arrangements may be taken into consideration
before starting a stereology analysis. First, remove the user bias by
having the individual performing the analysis blinded to the samples
and treatments represented on the slides. If the difference between
the two groups appears to be large, different sampling parameters
may be used for each group to increase effi ciency. Furthermore, an
independent second person should run the stereological analysis
using identical procedures in order to reduce personal bias and
increase validity. If inter-rater variability turns out to be high, further
sampling is needed if the users are both properly trained. If inter-
rater variability is low, this defi nitely lends confi dence to the valid-
ity of the results.
Performing many common stereology protocols, such as the
Optical Fractionator, using immunostained or counterstained
tissues, it is suggested that the user confi rm the depth of penetra-
tion and measure the fi nal mounted section thickness. While the
fi nal mounted and original section thickness are not required in
Stereo Investigator, if the original cut thickness is supplied, the
software can estimate the volume of the ROI as a bonus. A pilot
test of 5-6 sections may be measured as described below. This can
be easily accomplished within the Stereo Investigator software by
following the Focus Position Meter. First, focus at the top of the
section, where the cells fi rst start to come into focus. Move down
through the section following the Focus Position Meter to the
point where the staining starts to fade or is less clear; if focusing
down further does not bring more cells into focus, that point is the