disease is tied to taupathies Tau, a microtubule protein that is
hyperphosphorylated and also goes through conformational
changes where it makes it bind to tubulin.
This causes aggregation and production of neurofibrillary
tangles. Normal Tau consist of 2-3 mol of phosphate per ole of proteins.
The hyperphosphorylated tau can have 7-10 mol of phosphates per
mole of proteins. Alzheimer diseases begins in the hippocampus
and progress in
a definite shape, implying a connection of the
disease. Scientific studies verified that Hyperphosphorylted
Tau will promotes neurodegeneration with the mix of phosphorylation
at Thr 212, Thr 231 and Ser 262 which all produce
toxic consequences to the cell.
our research we utilized the model of Mouse to look
into the process of Tau toxicity caused by phosphorylation.
Fixation is technique we accustomed to maintain the
cell of the mouse in a like life state and
was completed immediately to avoid autolysis; we
applied methanol and glutaraldehyde. Transfection in the other hand is an
approach we used as well to introduce the molecular materials to the recipient
cell. We used the confocal microscopy to obtain the images of our slides
that indicate that gives a clear images of the cells and provides us
with nuclear and cytoplasmic
details. Ultimately our outcomes from the
images display did show what it expected to appear
like under standard conditions which might be
throughout error and or poor transfection efficiency.
Tau is a
neuronal microtubule integrated proteins that play
significant role in setting up cell polarity by stabilizing axonal
microtubules that function as a paths for motor protein
transportation process. In this
laboratory experiment, we are looking understand and work with the Tau protein.
Normal Tau and tubulin will form microtubules. The tau protein functions to
stabilize microtubules and is abundant in the central nervous system. It is
stated that there maybe be some clinical relation between the tau protein and
Alzheimer’s disease. Excessive or abnormally phosphorylated tau results in
PHF-tau and AD P-tau. These mutations and hyper-phosphorylation of tau inhibit
microtubule assembly and the microtubules are what keep neurons safe and allow
information to travel. Normal tau and tubulin will form microtubules, but
normal Tau and abnormal tau plus tubulin will slow the formation of
microtubules as concentration of abnormal tau increases.
10 mL of culture for 5 minutes, then we discard the supernatant.
pellet with 250 µL of Cell re-suspension Solution.
added a 250 µL of Cell lysis Solution and invert four times for mixing.
added 10 µL of Alkaline Protease Solution and invert four times to mix; then we
incubate it for 5 minutes at room temperature.
added a 350 µL of Neutralization Solution.
centrifuge at top speed for 10 minutes.
decant the lysate into Spin column.
then centrifuge at top speed for 1 minute at room temperature; then discard to
added a 750 µL of ethanol as a wash Solution, we centrifuge at for 1 minute and
discard to collection Tube.
transfer Spin Column and sterile 1.5mL micro-centrifuge tube to let it dry.
added a 100 µL of Nuclease-Free Water to the Spin Column.
we discard Spin Column.
measure DNA concentrations of A260/A280 ratio.
· Finally, we stored the DNA at -20 ?C.
Fixation: in fixation method we
used two fixative methanol and Glutaraldehyde
We aspirate, add
100 percent of methanol at -20 ?C.
We incubate it
in -20 ?C freezer for five minutes.
We wash with 1X
PBS for five minutes and we did this for three times.
We finally Store
it freezer at -20 ?C.
We aspirate and
add 1X PBS for five minutes.
We then aspirate
and add PEM buffer
We aspirate and
add 0.3 % of glutaraldehyde, 0.5 % Tween in PEM.
We incubate at
37 ?C for 10 minutes.
again and added a sodium borohydride about 10 mg/mL in 1X PBS and incubate for
We aspirate and added
a 0.1 M glycine in 1X PBS and it incubate for 20 minutes.
aspirate and added 1X PBS and let store in the fridge.
transfection method we used Transient technique because we wanted the cell to
exit only for limited time and not integrated into a genome.
We added a 25 µL
of Lipofectamine Solution to the 25 µL Plasmid Solution to make a 50 µL mixture
for each corresponding well, we then wait for 20 min.
added a 50 µL of Opti-MEM to each mixture to make 100 µL mixtures.
We aspirate the media
from wells and added a 100 µL of the mixture to the corresponding well.
We incubate for
We aspirate an added
a 200 µL of DMEM to each well
Finally, we incubate
Homogenization the brain: Homogenization is defined at reducing the size of
the particles, so that the material is uniform.
We started by
Prepare a100 mL of buffer of 100 mM MES at pH6.7, 1 mM MgCl2, and 1
We pour 50 mL of
buffer into a clean beaker, and place the beaker in ice.
We extract and
place a six mouse brains into the beaker that has the buffer.
We place the
brains into a homogenizer in ice.
We Added 2 mL of
clean buffer and start homogenize.
We transfer the homogenate
into two centrifuge tubes.
We determine mass
of each tube
the tubes at 100,000 x g under vacuum at 5 ?C for 40 minutes.
We then place
supernatant from the two tubes into a new tube.
We Make 40 %
glycerol solution with the supernatant.
We added 120 µL
of 1 mM GTP in Tris.
the solution into two centrifuge tubes.
We warm the
tubes in a 37 ?C water bath for half an hour
We then centrifuge
the two tubes at 20,000 x g under vacuum at 35 ?C for 20 minutes.
supernatant, and kept the pellet.
We used a 200 µL
of buffer to re-suspend cell pellet; and Place it in ice for 20 minutes.
We Spin it at 100,000 x g under vacuum, at 5 ?C for 10
minutes, we kept the supernatant
We added a 2 µL
of 1mM GTP in Tris to the supernatant.
Finally we incubate
in a 37 ?C water bath for 15 minutes
cells then skip this step. For glutaraldehyde cells we aspirate and permeabilize
0.2 % Triton, 5 % of donkey serum in 1X PBS for 10 minutes.
We aspirate and
block in 5 % of donkey serum in 1X PBS for one hour.
We aspirate and
incubate with 150 µL of primary antibody in blocking solution for overnight at
We aspirate, and
then added 1X PBS for five times for with five minutes of each.
We aspirate then
incubate with 150 µL of secondary antibody in blocking solution for one hour.
Finally we aspirate,
and added 1X PBS three times for 5 minutes for each.
Muscarinic Receptors Immunostaining
We make a 2 % NFDM solution in 3 mL of 1X PBS.
We make a 10 %
NGS in 1.0 mL of 2 % NFDM solution, and added 1 drop of Triton X to make the
We aspirate, and
added 125 µL of blocking solution to each well and incubate at room temperature
for 1 hour.
We make 1 mL
solution of 2 % NGS in 2 % NFDM for the primary antibody.
We dilute the
primary antibody in the 2 % NGS in 2 % NFDM solution.
We block, and aspirate,
then wash with 1X PBS three times for 10 minutes for each.
then added a 125 µL of primary antibody solution to the wells,
overnight in at 4 ?C.
We aspirate and
wash it with 1X PBS five times for 5 min for each.
We used 1 mL of
2 % NGS in 2 % NFDM for the secondary antibody solution.
We use a 1:1000
dilution for secondary antibody in the 2 % NGS in 2 % NFDM solution.
We aspirate, and
added 125 µL of secondary antibody solution to the wells.
We incubate for
1 hour at room temperature.
again and then wash with 1X PBS three times for 5 minutes for each.
We mount by
using vectashield mounting media with DAPI.
Finally, we store it
in 4 ?C
confocal microscope generates clear imagery of a specimen which would normally
look blurred when seen with a normal microscope. This really is accomplished by
eliminating most of the lights from the specimen that is not from the
microscope’s main plane. The image features less haze and much better contrast
than that of a normal microscope moreover shows a thin cross-section of the
specimen. Therefore, besides enabling better observation of fine details it is
easy to build 3D reconstructions of a volume of the specimen by organizing a
series of thin slices taken along the vertical axis
of energy found in the electron microscope is a beam of
electrons. Because the beam possesses a
remarkably short wavelength, it hits the majority
of things in it is way and then increases
the image resolution of the microscope drastically. The
electrons go out in a vacuum to stay away from contact with
deflecting air molecules, as well as magnets focus the beam on
the item to be seen. There are two types of Electron microscope;
TEM which is a beam of electron goes into the specimen and form white and black
image, it also have limited resolution. While SEM, scan the surface of the
image by using high energy of electron to generate different signals at the
It turned out to be that 48
hours of incubation was competent for
transfecting HEK-293 cells. A mutated NLS would not constantly block the nuclear localization of the various
forms of tau. Most likely there is certainly a difference between various mutations of the NLS sequence and nuclear localization of the forms of tau .Staining for ?-tubulin did
not really make the unique tubule structures in nearly all pictures.
Not even wild type tau transfect- ants revealed these types of structures. Which is maybe as a result of just how the tissues were taken
care of throughout the fixation and also staining processes. The methanol fixed R406W tau tissues were the exclusively tissues to display tubule structures. Muscarinic
receptor staining was very high for all those non-wild type forms of tau, except for PH NLS 48 hours. This tends to show that
the outcomes of PH tau on
muscarinic receptors are mediated by nuclear localization of the abnormal form of tau. Yet the muscarinic receptor staining within
this research failed to go
with the prior work conducted in visualizing muscarinic receptors.
Results from the images show that methanol cells
were didn’t have ? tubulin structure compared to glutaraldehyde
that show it does have it. Both glutaraldehyde and methanol had GFP signals in
tissue structures. We also learned that phosphatase inhibitor didn’t eliminate
the nuclear localization of R406W NLS tau and R406W tau. The level of transfection efficiency in
methanol was higher than in glutaraldehyde. We came to conclusion that the 48
hours transfection is more efficient than the 24 hours due to the fact it show
a clear vision if the cell we toxic and or fade away.
PCNA-1 also known as proliferating cellular
nuclear antigen 1; is a protein of DNA polymerase delta and is involved in the
control of DNA replication by increasing the polymerase process during the
elongation od the leading strand. PCNA-1 is strongly associated with nuclear
region where DNA synthesis is occurring.
All of these vectors were previously tagged with
GFP in our research experiment . PH Tau or Pathological Human Tau (Abnormal
tau) –expect to see microtubules broken down, tau will look like little dots,
SOMETIMES goes into nucleus WT= Wild Type Tau (normal)—everything should look
normal under microscope NLS= Wild Type Tau (Nuclear Localization Signal has
been changed) (tau does not go into the nucleus) R406 W—Wild Type Tau with 406
nucleotide changed from R to W (makes tau easier to get phosphorylated)—WT tau
with mutation to get it phosphorylated (may get phosphorylated or not, meaning
may or may not go into nucleus) PH NLS Tau—PH sometimes goes into nucleus so
having an NLS attached to PH will make it react differently.
It is crucial to notice that the purpose of
these kinds of research were to encounter a new methods along with the utilization of superior microscopes. Besides the laboratory mistakes that can potentially resulted in imprecise outcomes, the methods along with the techniques were documented. This really is a very good place
to start, which may provide for the long term tests by directing back up into these types of experimentations and
also idea our expertise and strategies. These types of findings would
not accommodate a lot information to be obtained regarding the function of various types
of tau in Alzheimer’s disease. The treatments has to be precious to develop the imaging of crucial structures
to allow a much better comprehension of the disease job