Jan 15, 2024
Preparation of mouse tissue homogenates for RT-QuIC assay
- 1Duke University
Protocol Citation: Arpine Sokratian, andrew.west west 2024. Preparation of mouse tissue homogenates for RT-QuIC assay. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwd2w9lmk/v1
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 28, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 91538
Keywords: ASAPCRN, alpha-synuclein, tissue homogenation, rt-quic
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020527
Abstract
This protocol is designed for a standardized and efficient procedure to homogenize mouse tissue, conducive for RT-QuIC analysis. The process involves treating samples with PBS mixed with Triton-X100, followed by homogenization using a prob-tip sonicator. Centrifugation is then employed to remove detergent-insoluble fraction, optimizing the tissue for subsequent
analysis. The use of Triton-X100, a mild detergent, ensures the release of alpha-synuclein aggregates, enhancing signal specificity without compromising structural integrity. The protocol's significance lies in addressing the critical need for a reliable method in preparing mouse tissue for RT-QuIC analysis, with the distinct signal generated in assays validating its efficacy.
Protocol materials
Low Protein Binding Collection Tubes (1.5 mL)Thermo FisherCatalog #90411
Step 1
Roche PhosSTOP EasyPackMerck MilliporeSigma (Sigma-Aldrich)Catalog #4906837001
Step 4
Roche cOmplete Tablets EDTA-free, EasyPackMerck MilliporeSigma (Sigma-Aldrich)Catalog #4693132001
Step 4
Protein LoBind Tubes, 1.5 mLEppendorfCatalog #0030108116
In 2 steps
Before start
The tissues need to be perfused with fresh PBS and flash-frozen in liquid nitrogen
Measure the weight of each eppendorf tube using analytical scale and label the tube with assigned number
Protein LoBind Tubes, 1.5 mLVWR InternationalCatalog #0030108116
Low Protein Binding Collection Tubes (1.5 mL)VWR InternationalCatalog #90411
Cut a tissue slice (around 100-200 mg) with a disposable blade on a plastic plate lid wrapped in a foil (all on ice), transfer to the Eppendorf protein low-binding tube (with known weight)
Safety information
biohazard; place disposable blades into biohazard needle container; material should be thrown away into biohazard bags previously placed into tube or a 15 mL falcon (secondary container)
Measure the weight of the tissue piece in a tube using analytical scales
Subtract weight of the tube to obtain the final weight of the brain slice (indicate the weight in the table)
Add 20 vol weight/volume of PBS buffer containing 1%Tx100 and protease/phosphatase inhibitors (Roche PhosSTOP EasyPackMerck MilliporeSigma (Sigma-Aldrich)Catalog #4906837001 ,Roche cOmplete Tablets EDTA-free, EasyPackMerck MilliporeSigma (Sigma-Aldrich)Catalog #4693132001 (for example, 100 mg – 5 mL) - transfer to 15 mL falcon tube;
Sonicate tissue samples for 00:01:00 , 5 sec ON and 15 sec OFF (On ice );
Use 0.16 inch microtip for homogenization
Equipment
Fisherbrand Model 120 Sonic Dismembrator
NAME
Sonicator
TYPE
Fisherbrand
BRAND
FB120110
SKU
LINK
Equipped with 1/8" probe
SPECIFICATIONS
1m
Spin down sonicated samples at 10.000 x g , 4 °C , 00:10:00
10m
Transfer supernatant into a new 15 mL falcon tube;
Aliquot the samples in lobind eppendorfProtein LoBind Tubes, 1.5 mLVWR InternationalCatalog #0030108116 tubes and store at -70 °C