Thiazole Orange is a dye that can be used for reticulocyte analysis.

Instructions
I. Solution preparation
1. Stock solution: Dissolve Thiazole Orange in DMSO or ethanol to prepare a stock solution with a concentration of 13 mg/ml. Since the dye is light-sensitive, it needs to be handled and stored in the dark.
2. Working solution: Dilute the stock solution to the working concentration according to the experimental requirements, usually 1 mg/ml. Use an appropriate buffer (such as PBS) for dilution.
II. Operation steps
1. Coupling with oligonucleotides:
(1) Thiazole Orange can be covalently coupled to modified oligonucleotides or linkers through amino-reactive functional groups (such as NHS esters or isothiocyanates).
(2) After coupling, purify the labeled oligonucleotides by high-performance liquid chromatography (HPLC) or gel electrophoresis.
(3) Verify the coupling efficiency by absorption and fluorescence measurements.
2. Reticulocyte analysis:
(1) Prepare a red blood cell suspension (prepared in PBS or isotonic buffer).
(2) Add Thiazole Orange at a working concentration (usually 1 mg/ml) and incubate at 37°C for 60 minutes to allow it to bind to RNA in reticulocytes.
(3) Analyze the stained cells using a flow cytometer or fluorescence microscope, selecting appropriate excitation wavelengths (~510 nm) and emission wavelengths (~530 nm).
3. Application of fluorescent hybridization probes:
(1) Use labeled oligonucleotide probes to detect specific nucleic acid sequences in hybridization experiments.
(2) Optimize hybridization conditions (such as temperature and buffer composition) to ensure specific binding of the probe to the target sequence.
(3) Measure the fluorescent signal using a fluorometer or fluorescence imaging system.
Notes:
1. Photosensitivity: Thiazole Orange is light-sensitive and should be avoided from prolonged exposure to strong light when used.
2. Storage conditions: The stock solution should be stored in a sealed container at -20°C to reduce moisture exposure and prevent degradation. Avoid repeated freezing and thawing.
3. Compatibility: Thiazole Orange binds strongly to nucleic acids. Avoid using solutions containing detergents or high salt concentrations, which may reduce its interaction with nucleic acids.