DNTPH

Guidelines (The following is a recommended experimental protocol for guidance only, and adjustments should be made based on your specific needs)

1. Stock Solution Preparation
1.1 Solvent: In published literature, DMSO is used to prepare DNTPH dye solutions for fluorescence detection.
1.2 Concentration Recommendation: It is generally recommended to prepare a high-concentration stock solution of 1-10 mM.

2. Working Solution Preparation
2.1 Diluent: Serum-free medium or PBS is typically used.
2.2 Working Concentration: 1 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use the solution immediately.

3. Staining Procedure
3.1 Sample Type: Prepare samples of Aβ42 fibrils or Aβ42 oligomers at different aggregation stages, then incubate with the DNTPH working solution for detection of the fluorescence signal of Aβ42 aggregates.
3.2 Incubation Conditions: A stable fluorescence response is achieved approximately 30 min after incubation of DNTPH with Aβ42 fibrils; light-protected operation is recommended during the experiment.
3.3 Washing Step: No specific washing step is required.

4. Control Setup
4.1 Negative Control: A DNTPH sample without Aβ42 fibrils, used to evaluate the background fluorescence of DNTPH.
4.2 Competitive Control: Add competitive molecules such as amino acids, enzymes and polypeptides, used to evaluate the selectivity of DNTPH for Aβ42 fibrils.
4.3 Positive Control: ThT is used for comparison in fluorescence detection of Aβ42 fibrils.

5. Detection and Analysis
5.1 Instrument Type: Fluorescence spectrophotometer, fluorescence microscope, confocal microscope.
5.2 Excitation/Emission Wavelength
5.2.1 The emission peak of the free probe is 708 nm, while the emission peak after binding to Aβ fibrils is 710 nm.
5.2.2 When DNTPH is used to detect Aβ42 oligomers at different aggregation stages, the emission wavelengths for the monomer aggregation stage (PM), primary nucleation stage (PP), secondary nucleation stage (PS) and protofibril formation stage (PF) are 657 nm, 639 nm, 630 nm and 648 nm, respectively.
5.3 Result Analysis
5.3.1 Fluorescence Intensity Change: DNTPH shows weak fluorescence in the free state, and produces a significant near-infrared fluorescence enhancement signal after binding to Aβ42 fibrils, with the maximum fluorescence intensity increased by approximately 18.1 times; the fluorescence response reaches a plateau about 30 min after incubation of DNTPH with Aβ42 fibrils.
5.3.2 Fluorescence Localization: DNTPH binds to Aβ42 fibrils and generates an enhanced fluorescence signal through the aggregation-induced emission (AIE) mechanism; in Aβ42 oligomer detection, DNTPH binds to Aβ42 oligomers at different aggregation stages and produces stage-related fluorescence responses.
5.3.3 Color Change: DNTPH produces a red near-infrared fluorescence emission signal after binding to Aβ42 fibrils.
After interacting with Aβ42 fibrils, DNTPH (1 μM) exhibits an approximately 18.1-fold fluorescence enhancement, and the fluorescence response reaches a plateau after 30 min; the binding affinity Kd of DNTPH for Aβ42 fibrils is 143 nM^[1].
DNTPH can detect the four aggregation stages of Aβ42 oligomers, including monomeric aggregation (PM), primary nucleation (PP), secondary nucleation (PS) and fibril stage (PF). DNTPH generates fluorescence signals at 657 nm, 639 nm, 630 nm and 648 nm in the above-mentioned stages, respectively.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.