Urine protein markers for the diagnosis of Parkinson's disease
A Parkinson's disease, urine technology, applied in the field of clinical diagnosis, can solve problems such as difficult to obtain
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Embodiment 1
[0018] Example 1 Rotarod Behavioral Detection of α-Syn(A53T) Positive Transgenic Mice
[0019] 4-week-old clean-grade male α-Syn(A53T)-positive transgenic mice [C57BL / 6J-Tg(PDGF-α-synuclein A53T) ILAS] (n=4) were purchased from the Institute of Medical Experimental Animals, Chinese Academy of Medical Sciences, The animal license is SCXK (Beijing) 2014-0011. Animal experiments followed review and approval (Animal Welfare Assurance Number: ACUC-A02-2014-007). All animals were kept in a standard environment with a 12-hour light-dark cycle (room temperature 22±1° C., humidity 65%-70%).
[0020] The rotarod behavior test was used to evaluate the motor coordination ability of the mice limbs. The rotational speed of the rotarod instrument (developed by the Institute of Materia Medica, Chinese Academy of Medical Sciences) was set to a constant speed of 25rpm, 30rpm, and 35rpm, respectively, with an interval of 30min and a time limit of 2min. The time when the mouse first fell off t...
Embodiment 2
[0026] Example 2 Immunohistochemical staining of α-Syn (A53T) positive transgenic mice
[0027] Immunohistochemical staining was performed after the last behavioral examination. Male C57BL / 6 mice of the same age were used as controls (n=3).
[0028] After the mice were anesthetized, the mice were perfused with normal saline and 4% paraformaldehyde successively, and the isolated brain was fixed in 4% paraformaldehyde for 24h, and then transferred to 4% paraformaldehyde containing 30% sucrose Dehydrate in the solid until the brain tissue sinks to the bottom. The brain tissue was removed, dehydrated, embedded and sectioned with a thickness of 4 μm. After dewaxing to water, the slices were repaired by microwave in EDTA buffer, washed with PBS buffer and then placed in 3% hydrogen peroxide solution, incubated at room temperature for 10 min, washed with PBS buffer, blocked with 5% BSA for 20 min, and then mixed with TH (1:100) antibody was incubated overnight at 4°C, and the seco...
Embodiment 3
[0030] Embodiment 3 urine collection and sample preparation
[0031] Urine samples of A53T mice were collected at the 2nd, 4th, 6th, 8th, and 10th month respectively, and the urine samples collected at the 2nd month were used as the control group. Mice were individually placed in metabolic cages overnight (12h) to collect urine samples. During the urine collection, no food was provided and water was freely available to avoid urine contamination.
[0032] The collected urine was centrifuged at 4°C and 3000×g for 10 min to remove cells and pellets, and the supernatant was stored at -80°C. Before urine protein extraction, the urine samples were centrifuged at 12000×g for 10 min at 4°C to remove cell debris. The supernatant was precipitated at 4° C. for 12 h using four volumes of pre-cooled ethanol. The above samples were centrifuged at 4°C and 12000×g for 10 min. The pellet was resuspended in lysate (8 mol / L urea, 2 mol / L thiourea, 25 mmol / L DTT and 50 mmol / L Tris). Protein ...
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