Detection of Disease-associated α-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated α-synuclein

The overall goal of the following experiment is to monitor the accumulation of the disease associated alpha synuclein in a transgenic mouse model of Parkinson's disease using a quantitative Eliza approach. This is accomplished by first allowing un inoculated mice to age normally or by inoculating mice intra cerebrally with a brain extract from sick M 83. After the mice develop clinical disease and are sacrificed, brain regions from the mice are dissected.

Next homogenous from each brain region are prepared in high salt buffer, and Eliza is performed on the homogenous in order to quantify the disease associated alpha-synuclein in each brain region. The Eliza results show that the disease associated form of the alpha synuclein protein is mainly accumulating in the coddle brain regions, including the mesencephalon and the brainstem, as well as in the spinal cords of the mice. The main advantage of this Eliza technique compared to previously available methods like western blood or immunohistochemistry, is that it provides rapid and reliable quantification of the disease associated alpha scle.

In this experimental model, This method can help monitor the accumulation and the propagation of this protein in the central nervous system of this MA three mice. That suggests involvement of a brain-like mechanism up to the development of the clinical science. Further developments of this method could be considered in the future for the study of various biological samples such as the cerebral spinal fluid or the blood that possibly also contain altered alphas forms demonstrating this procedure will be a technician from our laboratory.

All the procedures and protocols involving animals were in accordance with EC directive 86 6 0 9 EEC, and ratified by comm ETH, the French National Committee. For Consideration of Ethics and Animal Experimentation, the animals were housed and cared for in A and S'S approved experimental facilities in Leon Young after euthanizing mice and retrieving the brain's and spinal cord. According to the text protocol, place a whole brain in a Petri dish on ice under low power magnification.

Begin by separating one of the two olfactory bulbs by placing forceps just behind the bulb. Using a downward motion, detach it from the brain, then remove the second bulb in a similar manner. Now while using forceps to keep the brain in place, gently wedge a second pair forceps between the left and right cortices and move them forwards to dissociate the two halves.

Now continuing to hold the brain in place. Position the second forceps, two millimeters behind the cortex, and maintain the pressure upwards to lift the cortex off the hippocampus. Then peel off the first part of the cortex by starting at the hippocampus and moving towards the front of the brain.

Repeat with the second half. Next, position the open forceps around one of the hippo campi. Then gently close the forceps at the bottom of the organ and gently remove it.

Repeat with the second hippocampus. Position the forceps behind one of the striata and gently separate it from the brain. Use the forceps to remove any remaining cortex from the striatum before removing the second satu to separate the cerebellum from the rest of the brain with the forceps, gently depress the contour of the cerebellum.

Then place the forceps behind the cerebellum and move the forceps forward to remove it using the wide portion of the forceps raised them as cephalon, which is composed of four round structures to clearly see where a joins the brainstem. Then make a vertical incision at the junction before removing the brainstem position the forceps behind the mesencephalon and in size vertically to completely separate it from the remaining brain. To prepare brain homogenous, add an adequate volume of HS buffer to the dissected brain regions to reach an expected percent homogenate as shown in the table below.

Using a tissue grinder composed of bo silicate glass tube and tube pests A and B, pour a brain region to be crushed into the tube. And then with pestle a homogenize the tissue 10 times to dissociate it. Then switch to pestle B and homogenize an additional 20 times.

With a transfer pipette, transfer the homogenate into a 1.5 milliliter tube, repeating the homogenization with the remaining brain samples. Centrifuge the samples at 1000 G for five minutes at four degrees Celsius, and then collect the snat and divide them into 200 microliter Ali Watts. Store at minus 80 degrees Celsius for subsequent Eliza analysis to carry out Eliza on brain homogenous.

Begin by using 50 micromolar carbonate buffer to dilute the coating anti alpha synuclein, rabbit polyclonal or monoclonal clone 42 antibody to 0.01 nanograms per milliliter with 100 microliters of the antibody coating solution coat the wells of 96. Well microplate and incubate at four degrees Celsius overnight the following day. Add 300 microliters of PBST to the wells and use a plate washer to wash the plates five times while working at room temperature.

Moving forward, add 200 microliters of T 20 PBS blocking buffer to each well and shake the plates at 150 RPM for one hour before using PBST to wash the plates five times using PBST 1%BSA dilute the brain homogenous using the guidelines listed here and add 100 microliters to each. Well then incubate at room temperature and 150 RPM for two hours before using PBST to wash the plates. Five times after the final wash, add the necessary alpha synuclein detection antibodies diluted in PBS BST and 1%BSA as listed in table two of the text protocol incubate at room temperature and 150 RPM for one hour before washing the plates five times as before.

Next, add either anti mouse or anti rabbit IgG HRP conjugates diluted to one to 8, 000 in PBST 1%BSA and incubate at 150 RPM for one hour before washing the plates five times. Then add 100 microliters of TMB solution to each well and incubate at 150 RPM for 15 minutes in the dark. Stop the reaction by adding 100 microliters of one normal hydrochloric acid per well.

Then use a microplate reader to measure the absorbance at 450 nanometers for data analysis. Subtract the optical density value obtained in the blank. Well containing only buffers and reagents from the optical density.

Values determined from brain samples carry out statistical analysis according to the text protocol in this study, the Eliza's performed specifically identified disease associated pser 1 29 alpha synuclein in brain homogenous from old sick M 83. Mice not present in young M 83 mice. Additional antibody tested like LB 5 0 9 showed strong signals in sick but not healthy mice.

The analytical sensitivity of the ELIZA described in this video was compared to a previously described western blot method. Approximately 10 micrograms of brain equivalents were sufficient to produce a positive P or 1 29 alpha synuclein Eliza signal from a sick mouse. However, at least 200 micrograms of brain sample were needed to detect pser 1 29 alpha synuclein by western blot in sick and old M 83 mice brain homogenous from the mesencephalon brainstem and spinal cord showed marked immunoreactivity with both LB 5 0 9 and PSER 1 29 antibodies.

And the eliza, however, no signal was detected in the other brain regions tested the immunoreactivity in the different brain regions of M 83 mice developing an accelerated clinical disease following the injection of a brain extract from a sick M 83 mouse was indistinguishable from that seen in aged UN inoculated M 83 mice. These results are fully consistent with those obtained by Western blot and immunohistochemistry showing a much greater deposition of pser 1 29 alpha-synuclein in the codal regions of the brain and spinal cord. Once this section of the brain has been achieved, the modernization of sample is a rather long and crucial step.

It requires the use of high salt buffer to correctly identify the mineral reactivity of a disease associated fain clinic. The Eliza tells bad follow and peripheral in a few hours. This procedure demonstrates that the disease associated protein is mainly accumulating in the co regions of the brain and in the spinal cord as previously observed using western blood or immunohistochemistry.

This new eza method pa, the way for researches in the field of sin nuclear neuropathies to explore the propagation of the disease associated alphas cle. Since a precise quantification of the protein can be obtained throughout the central nervous system of the mice, it'll be, for example, especially useful to follow the effects on the development of the pathology of a potential causal factor or the contrary of a possible therapy of Parkinson's disease.