JF629-HTL

Guidelines (The following is a recommended experimental protocol for guidance only; adjustments are required based on your specific needs)

1. Stock Solution Preparation
1.1 Solvent: DMSO.
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: Select an appropriate medium or buffer according to the HaloTag labeling system.
2.2 Working Concentration: 1-5 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare fresh immediately before use.

3. Staining Procedure
3.1 Sample Type Description
3.1.1 Adherent cultured rat hippocampal neurons
3.2 Incubation Conditions: Omitted
3.3 Washing Step: Wash to remove unbound dye after labeling to reduce background fluorescence.

4. Control Setup
4.1 Blank control to eliminate reagent fluorescence interference.

5. Detection and Analysis
5.1 Instrument Type: Fluorescence microscope, confocal microscope or live-cell imaging system.
5.2 Excitation/Emission Wavelength: Use a 639 nm laser for excitation and a 690/50 nm emission filter for detection.
5.3 Result Analysis
5.3.1 Fluorescence Intensity: Under 450 nm illumination, the fluorescence turn-on fold of the psHaloTag system reaches 6.7 times, and fluorescence reversibly recovers in dark conditions; when neurons receive electrical stimulation and generate action potentials, the fluorescence of the HaloCaMP calcium sensor increases (ΔF/F₀), with a larger ΔF/F₀ value under high-frequency action potential train stimulation; calculate the background-subtracted fluorescence intensity and normalized fluorescence change (ΔF/F₀).
5.3.2 Fluorescence Localization: Target-specific localization is achieved based on the fused psHaloTag1a vector (nuclear H2B, mitochondrial TOMM20, F-actin LifeAct); HaloCaMP is expressed in the soma and dendrites of cultured hippocampal neurons.
5.3.3 Color Change: Far-red fluorescence (~650-700 nm); increased fluorescence intensity indicates elevated Ca²⁺ concentration or activation of light-controlled labeling.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only. Further protocols information, click here.