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Lab
Research
Philosophy
The lab philosophy is to stimulate trainees to develop their
fullest potential as independent investigators while
establishing good networks and a collaborative spirit. To
facilitate their critical thinking and in depth analyses of
data the lab developed a tradition of two weekly meetings.
One is an informal gathering aimed at discussing relevant
new publications and updating each other on the major tasks
ahead and to resolve laboratory housekeeping issues. The
other meeting is aimed at discussing in depth laboratory raw
data, their significance in the research area and also aimed
at discussing methodological challenges and
“techniques-troubleshooting”.
The research is focused predominantly -but not exclusively-
on the “road maps” offered by the funded grant applications.
However, when novel and unexpected data provoke new
questions or new leads, these are considered as potential
new areas of research if deemed of importance.
As summarized above in
Collaborations, the lab is open to collaborations with
national or international laboratories where the cooperation
will increase the value of the research undertaken. These
collaborations are at large based on pre-existing
friendships and mutual trust. Whenever possible, the
exchange of trainees/collaborators is encouraged. It is
expected that trainees become experts in the performance of
several techniques employed in the lab and that they assist
each other in optimizing the application of these
techniques.
Within the lab we foster a spirit of openness and mutual
team support. Senior graduate students and postdoctoral
fellows contribute substantially to the development of the
junior trainees. Projects are often shared by more than one
member of the lab in order to efficiently complete research
tasks by the summation of complementary expertise.
Methodologies
The lab aims to investigate a scientific
problem or research question using a multidisciplinary
approach wherever possible. Thus, the lab has established a
variety of methodologies ranging from molecular approaches
to whole animal experimentations. The lab has created a
number of transgenic mouse models such as: a transgenic mice
line over-expressing NGF in the stem and spinal cord; a
transgenic mice line coded McGill-Thy1-APP mimicking the
AD-like amyloid pathology with excellent survival and
amyloid plaque formation starting 4 months of age; a rat
transgenic model coded UKUR-25 with a an early AD-like
pathology limited to the occurrence of pathological Aβ
pathology and more recently a rat transgenic model
displaying the full AD-like amyloid pathology with a single
transgenic construct of APP with the so-called Swedish and
Indiana mutations, carrying a single transgene per allele.
Despite of the low “transgene dose” these rats display early
cognitive impairments, accumulation of Aβ peptides, amyloid
plaques, CNS inflammation and dystrophic neurites.
The breeding and genotyping of these models
is carried out in our lab. The laboratory uses a great deal
of tissue culture experimentation with established cell
lines and primary cultures of neurons and glia. These cells
are occasionally transiently transfected and a number of
stably transfected cell lines have been generated by our
lab. Primaries cultures are often generated from dissociated
cells from the cerebral cortex, hippocampus or septal
nucleus.
Whenever necessary our lab generates
monoclonal antibodies (Mabs). Examples of such are McSA1
against Aβ peptide, McKA1 against the Glutamate transporter
1; TOH against Tyrosine hydroxylase, NC1/34 against
Substance P, DβH against Dopamine-β-Hydroxylase, P4C1
bi-specific antibody against substance and HRP, Y4C7
bi-specific antibody against Somatostatin and HRP. We also
produced the MAP (mouse anti-HRP antibody) and RAP (rat anti
–HRP antibody) that used in conjunction with a rabbit
antibody against rat Fc portion of IgGs (for RAP) or a
rabbit antibody against the mouse Fc portion of IgGs (for
MAP) allows a clean amplification of the signal enabling the
detection of low amounts of antigens.
The laboratory has a long tradition of
optimizing immunohistochemical approaches for light and
electron microscopy. Many of the protocols developed in the
Cuello lab h are now being used widely in many labs . These
immunocytochemical procedures and novel applications of the
hybridoma technology are described in detail in
corresponding chapters of the past best-seller manuals
“Immunohistochemistry” (1983) and Immunohistochemistry II
(1993) both Edited by A. C. Cuello and published by John
Wiley NY. The lab uses a great deal of light, confocal and
electron microscopy approaches often in collaboration with
the Ribeiro-da-Silva lab. When necessary the lab resorts to
micro-dissection of CNS nuclei to specific neurochemical
investigations (for description of methods see
”Micro-dissection Techniques” (1983), John Wiley and Sons,
ed. A.C. Cuello.)
An important component of the studies is the
regular application of neurochemical procedures to
investigate important CNS markers. Typically, there are
relevant proteins such as neurotransmitter markers, enzymes
and growth factors. The methods applied are Western
blotting, dot blots, immunoprecipitation, zymography and
occasionally radiochemical approaches to measure enzymatic
activity.
For whole animal experiments drugs are often
applied systemically or into the CNS. In the latter case by
using cannulae permanently implanted in key areas of the
brain and connected with an osmotic mini-pump for the steady
and regulated delivery of compounds.
The lab has contributed to install a Faculty
of Medicine Animal behavior facility for the testing of the
cognitive status of mice and rat as a consequence of drug
treatment, transgenesis or both. The most regular technique
is the use of the so-called Morris Water Maze for the
testing learning and memory. The lab also applies protocols
for Novel Object Recognition and Location and Fear
Conditioning Techniques.
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