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started by Marlin Dickson on 29 May 12
started by Marlin Dickson on 29 May 12
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started by Marlin Dickson on 29 May 12
new location of the escape platform. If this experimental subjects had
not found the platform when the allowed time-lapse was over, they
were guided to it. Once on the platform, rats were permitted to stay
for 5 seconds to let them take notice of the location of the platform. Eight
acquisition trials were performed for any rat, with a 5-min period of time
between each one. Plenty of time latency for reaching your platform was
measured.
2. 5. Experiment 2-effects of prolame treatment relating to the number and
morphology associated with nNOS expressing cells together with dendritic spine densities involving
projection neurons in the hippocampus
After performing the MWM, five rats with each group were
aimlessly chosen for histological reviews, excepting the GI party in
which the selection was according to observation from vaginal smudges -
only rats inside proestrous stage were chosen. It is worth leading
out that the proestrus with female rats represents the greatest estrogen
blood levels seen during estral cycle [42] [43]. We chose this criterion
to make an equivalent comparison along with the other two treatment communities
and also tominimize the intragroupal variability of measured histological
parameters, which in accordance with the literature [44, 45], rely critically
on the intracerebral estrogen concentrations. Vaginal smears obtainedwith cotton
swabs were stained applying Papanicolau staining procedure. The presence
of nucleated epithelial cells in the vaginal smear was used being a criterion
for proestrus determination [46].
For perfusion-fixation, rats were anesthetized with the overdose
of sodium pentobarbital (Sedalpharma, México, South america, 63 mg/kg
b. w., i. p.) and transcardially perfused with 0. 9% saline, implemented
by ice-cold fixative that contains 4% paraformaldehyde dissolved
with 0. 1 M phosphate stream (PB, pH 7. 4), for 15 min. Brains were
removed, totally rinsed in PB together with blocked sagitally in two
halves, for both nNOS immunohistochemistry together with Golgi-Cox
impregnation.
One 50 % of each rat brainwas raised for nNOS immunohistochemistry.
The brain tissue containing dorsal hippocampus has been coronally sliced
using some sort of Leica vibratome (VT1000). The thickness in the section was
50 μm. One of the many 4 sections from Bregma â2. 40 mm to
â4. 00 mm [47] (8 areas per rat) were processed for nNOS freefloating
immunoreaction. After corresponding
washings (3Ã10'), solar panels were further incubated using
secondary antibody donkey anti-mouse Alexa fluor 488 (1: 1000,
Invitrogen, Life Technologies, Carlsbad, California, NORTH AMERICAN, in TBST
with 1% NHS), with 4 °C overnight. Sections were then thoroughly
rinsed and mounted with Vectashield (Vector Laboratories, Inc,
Burlingame, CA, USA).
For mobile counting, eight slices with each rat were applied at 150 μm
span. Two independent researchers who have been blind to the experimental
conditions carried out mobile counting. Principal neurons
situated CA1, CA3 and dentate gyrus (DG), immunoreactive to help
nNOS were counted which has a modified method from [48, 49]. Nikon
ECLIPSE 50i fluorescence microscope with 40Ã objective with B-2A
longpass emission filtering was used. Counting involving nNOS+cells was
performed under microscope, inside a duration of 540 μm (corresponded
to your diameter of the 40Ã objective field) of the cell
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