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Optical Fractionator - Fluorescence
The total number of stained cells (e.g. tyrosine hydroxylase positive, TH+, neurons) and
unstained cells (e.g. TH- neurons) in the region(s) of interest (e.g. substantia nigra pars
compacta and/or ventral tegmental area) were counted using the optical fractionator and
our previously described counting criteria (see references below). Briefly, neurons were
counted as TH+ if they showed: (a) TH immunoreactivity within the cell body, (b) part of
the nucleus of that cell was inside the counting frame without touching the avoidance
lines, and (c) a portion of the nucleolus within that nucleus was in focus between the top
and bottom boundaries of the counting frame or not in the guard zone. TH- neurons were
counted if (a) a neuronal nucleus was visualized without cytoplasmic staining, (b) the
nucleus must have a size equal to or greater than the diameter of the average TH+
nucleus, (c) it has a macronucleolus and more than one small nucleoli, (d) the shape of
the nucleus is round to oval, (e) the nuclear membrane typically smooth, (f) the nucleus
was partially or entirely inside the counting frame without touching the avoidance lines,
and (f) a portion of the nucleolus within that nucleus was in focus within the top and
bottom boundaries of the sampling frame, i.e. not in the guard zone.
The sections were ordered from rostral to caudal by visual inspection at low
power. The regions of interest were outlined at low magnification (4x objective) and
sampled at high magnification (100x oil immersion objective) using StereoInvestigator
(Microbrightfield, VT). We performed IHC using every 4th section from the midbrain, but
performed stereology on every 8th section using a random first section to start. This
provided two sets of sections should one set be uncountable due to a damaged or folded
section. We counted and report data from one side of the substantia nigra. We
determined our sampling criteria empirically based on initial testing and confirmation
with different experimental manipulations as previously reported. We counted enough
TH+ neurons to yield a Nest having a coefficient of error (C.E.)  10%. We accept a
larger C.E. for TH- neurons as they are more difficult to identify, fewer in number, and
not the primary purpose of counting. To achieve these we sampled every 8th section of
the SNpc and spaced our sampling frames every 200 µm (along the x-axis) and 100 µm
(along the y axis) from each other based on the shape of the region. We use a sampling
frame size of 70 µm (x-axis) x 50 µm (y-axis) which allows for a reasonable border using
our microscope with a 100x objective and our camera.
We used an Olympus Provis AX-70 microscope equipped with 4x dry and 100x
oil immersion objectives, a Heidenhain linear encoder mounted to a LEP motorized XYZ
controlled stage connected a MAC 5000 driver. An Optronics DEI-750 CE camera was
used for real time collection of video images and was connected to a Dell computer using
a frame grabber board.
For fluorescence detection the Olympus Provis AX-70 microscope was equipped
with the AX reflected light module (halogen lamp housing, mercury burner power
supply, collector lens, and reflected light filter slider), fluorescence filter cubes, and the
AX-REXBA excitation light balancer. We used the following commercial filters
available from Olympus (details of the dichroic mirror, exciter filter and barrier filter can
be found at http://www.olympusamerica.com/seg_section/uis2/seg_uis2_mirror.asp): (1)
U-MWU, UV range for DAPI, blue color, (2) U-MDA/FI, DAPI/FITC, for DAPI and
ALEXA fluor 488, blue and green, (3) U-MDA/TRITC, for DAPI and ALEXA fluor 594,
blue and red, (4) U-MDA/FI/TRITC, for DAPI, ALEXA fluor 488, and ALEXA fluor
594, blue, green, and red. The spectral characteristics of the commercial ALEXA
fluorophores can be obtained at
http://probes.invitrogen.com/handbook/sections/0103.html. A 25 ND filter was used in
the reflected light filter slider to reduce the intensity of the light source only with the
100x objective to reduce bleaching. An Optronics DEI-750 CE camera with keyboard to
adjust all camera settings was used. The only setting on the camera altered during
stereology was the gain (exposure) which was always reduced with the 100x objective
relative to use with the 4x objective.
References
Thiruchelvam, M., Richfield, E. K., Baggs, R. B., Tank, A. W., and Cory-Slechta, D. A.
(2000) J. Neuroscience 20, 9207-9214
Barlow, B. K. , Thiruchelvam, M., Richfield, E. K., and Cory-Slechta, D. A. (2004) Dev.
Neurobiology 26, 11-23
Thiruchelvam, M., Powers, J., Cory-Slechta, D. A., and and Richfield, E. K. (2004) Eur.
J. of Neuroscience 19, 845-854