I am trying to simulate AD conditions in rats using Amyloid Beta 1-42 oligomers for which I would like to know the best methods for its processing. I have read about the use of Hexafluoroisopropanol (HFIP) and Dimethyl Sulfoxide (DMSO). If any one has used these substances or any other better methods of Amyloid Beta 1-42 processing kindly let me know.
Hi Sairaj Satarker
I have never done it myself but have recently seen the poster at a meeting. In this publication, they are showing a method to generate the fibrils (1 week at 37°C shaking at 100rmp). Also, they describe that the fibrils form the brain differ from the ones made in vitro.
Best,
Oliver
Kollmer, M., Close, W., Funk, L. et al. Cryo-EM structure and polymorphism of Aβ amyloid fibrils purified from Alzheimer’s brain tissue. Nat Commun 10, 4760 (2019). https://doi.org/10.1038/s41467-019-12683-8
I tried the fluorescent labelled fibril Amyloid Beta 1-42 preparation in vitro. The peptide is difficult to dissolve in PBS directly. Try to use DMSO to dissolve the peptide first and then dilute with PBS. I tested using 20 μl DMSO to dissolve 0.1mg peptide and dilute with 166 μl PBS to make a 100 μM stock buffer. Then incubate the stock buffer 1 week at 37°C shaking at 100rmp.
Check the method in my article ( Article The Effect of Acori Graminei Rhizoma and Extract Fractions o...
)Intra-Hippocampal Injection
Aβ1-42 powder (ANASPEC, 500 μg) was dissolved in 250 μl sterile saline solution containing 1% NH 4 OH to make 2 μg/μl working solution. The solution was mixed and incubated at 37 ºC for 6 days, allowing Aβ aggregation in the
solution.
To inject the aggregated Aβ solution into the hippocampal CA1 area, a surgical procedure was performed on mouse anesthetized with 0.4% sodium pentobarbital anesthesia (0.02 ml/g b.w., i.p.). The animal’s scalp was
shaved and sterilized with iodine tincture and ethyl alcohol. The mouse was then immobilized onto a stereotaxic frame and a 1.0 cm midline sagittal incision was made to expose the skull. After a tiny hole was drilled into the skull, Aβ1-42 (4 μg/2μl) was injected into hippocampus CA1 area bilaterally at the coordinates 2.3 mm posterior to Bregma, 1.8 mm lateral to the midline, and 2.0 mm ventral to the brain surface according to the mouse brain atlas in
stereotaxic coordinates of Paxinos and Franklin using a 5 μl microsyringe (Hamilton) driven by a minipump (KDS model 310 plus, KD Scientific, Holliston, MA) at a rate of 0.2 μl/min. The needle was left in place for an additional 5
min for sufficient dispersion of the solution and then withdrawn slowly. The tiny hole was closed with bone wax (Ethicon). The scalp wound was treated with antibiotics to prevent bacterial infection, and then sutured. The mice in the
saline group underwent the same procedure except that sterile saline was injected with the same volume instead of Aβ solution. Naive (control) mice did not undergo any surgical procedure, nor receive any injection. Mice with an
incorrect injection site or no signs of learning impairment were excluded in the study.
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