The Stereology and 3D Volume Analyses in Nervous Tissue - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 38

Counting will be done at high power (e.g., ×40 or ×100).
First, the section thickness is measured, or section thickness can
be measured while counting (the latter is highly recommended).
Be sure to select Refocus to the top of each section at each grid
site and manually enter an average mounted section thickness
(Fig. 4c arrow). Your progress through each set-up stage may be
viewed at the left side of the screen; as each step is completed, a
check mark is placed next to it. Next, defi ne the size of the counting
frame (Fig. 4c ); typically 25 × 25 mm is used, but the size depends
on homogeneity of the cell distribution as well as the area selected.
The counting frame appears as a square or rectangle depending on
desired dimensions. The top and right sides are green, indicating
that you may count an object of interest on this line. A red line
delineates the left and bottom of the counting frame, indicating
that an object on these lines must not be counted. An optimal
gauge of sampling intensity, for the sake of effi ciency, is the pres-
ence of one to three objects present per counting frame ( 10 ).
The counting frame size and grid size (spacing between sampling
sites) will affect the number of sites that will be sampled in each
section. When fi rst defi ning the parameters of a study, the user
may specify an average target value of 10 or 15 (for example) for the
number of sampling sites per section in order to have the software
then determine an appropriate grid size. Then click estimate grid
size , which will show a bird's eye view of the ROI with all the
counting frames within it, using the automatically generated grid
size. Now select Disector Options - setting the height of the guard
zones. For example, if your processed tissue thickness is 20 mm
and the guard zones are 2 mm from the top and bottom , then the
total Disector height is 16 mm. Finally, select a marker for counting.
Keep in mind the size of the marker with respect to the objects
counted; if your objects are very small, such as vesicular trans-
porters, the marker should be of a commensurate size. With
the marker selected, count away! For each new section, focus on
the top where objects fi rst become clear; this will become “zero”
on the Focus Position Meter (Fig. 4d ) for each sampling site. When
the counting is fi nished for one animal, a results window of
Previous Stereological Runs will open; click on View Results . Stereo
Investigator will show the estimated total number of objects in the
ROI. Estimates of the total number of objects in a given region
may be calculated independently by multiplying the total number
of objects actually counted by the fraction of ROI that was actually
sampled (which is easily calculated since the section increment,
counting frame size, grid size, average mounted section thickness,
and disector height are all known).