Preparation of mouse tissue homogenates for RT-QuIC assay
Arpine Sokratian, andrew.west west
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.
Before start
The tissues need to be perfused with fresh PBS and flash-frozen in liquid nitrogen
Steps
Measure the weight of each eppendorf tube using analytical scale and label the tube with assigned number
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)
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 (
Sonicate tissue samples for 0h 1m 0s, 5 sec ON and 15 sec OFF (On ice);
Use 0.16 inch microtip for homogenization
Equipment
| Value | Label |
|---|---|
| Fisherbrand Model 120 Sonic Dismembrator | NAME |
| Sonicator | TYPE |
| Fisherbrand | BRAND |
| FB120110 | SKU |
| Equipped with 1/8" probe | SPECIFICATIONS |
Spin down sonicated samples at 10.000x g,0h 0m 0s, 4°C, 0h 10m 0s
Transfer supernatant into a new 15 mL falcon tube;
Aliquot the samples in lobind eppendorf-70°C
