Red-HyP-1 is a red dye that can be used for in vivo hypoxia imaging. The maximum emission maximum of Red-HyP-1 is 798 nm.

Under hypoxic conditions, CYP450 enzymes react with the N-oxide group, undergoing two-electron reduction to yield the uncapped dye (Red-HyP-1) which results in a red-shifted emission. HyP-1 has an absorbance and emission maxima at 670 and 697 nm, respectively, while red-Hyp-1 (the turned over product) has an absorbance and emission maxima at 760 and 798 nm. [1]

Instructions
1. Solution preparation
Stock solution: Dissolve Red-HyP-1 in anhydrous DMSO or DMF to prepare a 1–10 mM stock solution.
Working solution: Dilute to a working concentration of 1–10 µM using an experimental buffer (e.g., PBS, pH 7.4).
2. Experimental steps
In vitro experiments
1) Add Red-HyP-1 to the sample solution (e.g., cell culture medium or biological buffer).
2) Incubate for 30 minutes to 1 hour at 37°C, away from light.
3) Detect fluorescence signal:
Excitation wavelength: 720–750 nm
Emission wavelength: 798 nm
In vivo hypoxia imaging
1. Inject Red-HyP-1 into experimental animals (e.g., tail vein injection, the specific dose depends on the experimental design, usually 1–5 mg/kg).
Wait 30 minutes to 1 hour after injection and detect using a fluorescence imager.
2. Signal detection equipment
Use near-infrared fluorescence imaging system or fluorescence microscope to record the signal.
3. Calibration and control
1) Control experiment: Set up control groups with normal oxygen concentration (such as 20% O₂) and low oxygen concentration (such as 1% O₂). Verify whether the signal is related to hypoxia.
2) Standard curve: Use samples under known oxygen concentration gradient to establish the relationship curve between fluorescence signal and oxygen concentration.
Notes
1) Photosensitivity: Red-HyP-1 is sensitive to light. Avoid strong light exposure throughout the experiment to prevent fluorescence signal attenuation.
2) Storage conditions: Red-HyP-1 should be stored at -20°C in the dark and avoid repeated freezing and thawing.
3) Dissolution stability: Ensure that the solution is fully dissolved before use; if necessary, undissolved particles can be removed by low-speed centrifugation.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.