P6-Aggrate

Operating Instructions
Operating Instructions (The following is a recommended experimental protocol for guidance only, and adjustments are required based on your specific needs).

1. Stock Solution Preparation
1.1 Solvent: Most dyes are usually dissolved in organic solvents, such as anhydrous DMSO.
1.2 Concentration Recommendation: Prepare a high-concentration stock solution at 1-10 mM.

2. Working Solution Preparation
2.1 Diluent: Serum-free medium or PBS is generally used. Proteins and esterases in serum may interfere with staining results or cause dye hydrolysis.
2.2 Working Concentration: 4-50 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately.

3. Staining Procedure
3.1 Sample Type: Adherent cells (HeLa cells)^[1].
3.2 Incubation Conditions: Incubate cells with 5 μM P6 and MG132 (HY-13259) for 24 h; avoid light during confocal imaging.

4. Detection and Analysis
4.1 Instrument: Confocal fluorescence microscope
4.1.1 Excitation wavelength: 488 nm; emission wavelength can be adjusted within the range of 520-580 nm.
4.2 Result Analysis:
4.2.1 Fluorescence intensity change: Fluorescence intensity increases upon binding to amorphous aggregated proteomes; fluorescence intensity decreases and red shift occurs after aggregate clearance.
4.2.2 Fluorescence localization: Localizes to proteome aggregates (which can be small speckle-like particles or large perinuclear aggregates depending on the cell stress status).
4.2.3 Color change: Aggregates show green fluorescence under standard imaging conditions; a shorter emission wavelength (blue shift) indicates higher aggregate compactness, while a longer emission wavelength (red shift) indicates lower aggregate compactness.