The Stereology and 3D Volume Analyses in Nervous Tissue - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 36

bottom of the section. The depth traversed provides information
about the shrinkage of your tissue, a common occurrence for
immunostained and counterstained tissue that is dehydrated
through graded alcohol-xylene-based washes. Confi rmation of
depth of penetration and total section thickness is helpful prior to
starting stereological counting. It may be advisable for new stere-
ology users to learn how to measure the fi nal mounted section
thickness using differential interference contrast (DIC) microscopy
or other means using brightfi eld microscopy. For stereological
counting using brightfi eld, the user must use an objective lens with
a suffi ciently high numerical aperture (commonly 1.4 N.A.),
achieve Kohler illumination, and then open the condenser aperture
all the way, in order to get the thinnest possible depth of fi eld.
Without this step, the counting results will be far less robust than
expected. Guard zones are established at the top and bottom of the
section to allow for sampling in tissue where there are no “lost
caps” or “craters” and also avoids sampling from an area where the
tissue may have been badly deformed by the cutting process in a
number of different ways. The guard zone is determined empiri-
cally for each study.
Equipment: The essential equipment required for a stereology sys-
tem includes a microscope (e.g., a modern Leica, Zeiss, Nikon, or
Olympus) with a motorized x-y-z stage interfaced with a dedicated
cooled monochrome for fl uorescent work and a color camera for
brightfi eld work, and a microcator (Z focus position linear encoder)
with the stage controller connected to a PC (Intel® Quad Core
Xeon Processor W3540 2.93 GHz, 12 GB DDR3 SDRAM,
2 × 500 GB hard drives, 1 GB nVidia 9800 GT video card, DVD
drive, USB and serial ports, Windows 7, 64-bit operating system).
This confi guration is based on the current hardware needed for
Stereo Investigator . Other systems may have different hardware cri-
teria. Whichever stereology software is used, the ability to generate
a grid of sampling frames to overlay the microscopic images on the
monitor for individual signal counting is required.

5. Using the
Optical
Fractionator in
Stereo Investigator

Sequential procedures for counting using Stereo Investigator software
(MBF Bioscience) and the Optical Fractionator probe are detailed
below. Open the Stereo Investigator software and from the Probes
menu choose the Optical Fractionator Workfl ow . In the next window,
select Start a New Subject or Load subject from existing data fi le . At
the bottom of the Set up the Subject panel (Fig. 4a ), enter the fresh
tissue cutting thickness. When starting the fi rst section for a new subject,
select a reference point. This may be any point in the section or