PNO1

Operating Instructions (The following is a recommended experimental protocol for guidance only; adjust according to specific requirements)
1. Stock Solution Preparation
1.1 Solvent: DMSO.
1.2 Recommended concentration: 5 mM.
2. Working Solution Preparation
2.1 Diluent: 10 mM PBS, pH 7.4.
2.2 Working concentrations: 5 μM, 1 μM, 10 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately.
3. Staining Procedures
3.1 Sample Types^[1]:
3.1.1 Suspension cells: Primary alveolar macrophages.
3.1.2 Adherent cells: Primary lung fibroblasts.
3.1.3 Tissue samples: Paraffin-embedded mouse lung sections, paraffin-embedded human lung tissue sections.
3.2 Incubation Conditions:
3.2.1 Suspension and adherent cells: 1 μM PNO1, 15 min.
3.2.2 Tissue sections: 10 μM PNO1, 30 min.
3.3 Washing Steps:
3.3.1 Cells: Wash twice with PBS.
3.3.2 Tissue sections: Rinse three times with PBS before incubation; wash three times with PBS after incubation.
4. Control Setup
4.1 Set up a negative control probe (PNO3) for tissue staining.
4.2 Set up a pretreatment control group with NO scavenger (Carboxy-PTIO) for tissue staining.
5. Detection and Analysis
5.1 Instruments:
5.1.1 Fluorescence spectrophotometer: Ex = 545 nm, Em = 550-650 nm.
5.1.2 High-content imaging system: Ex = 520-550 nm.
5.1.3 Fluorescence microscope: Used for cell and tissue imaging.
5.2 Result Analysis:
5.2.1 Fluorescence intensity change: The fluorescence intensity of PNO1 increases in a dose-dependent manner with elevated NO concentrations; the fluorescence intensity of cells and tissues with pulmonary fibrosis (PF) lesions is significantly higher than that of the normal control group.
5.2.2 Fluorescence localization: Yellow fluorescence appears in PF-lesioned cells and lung tissue regions.
5.2.3 Color change: The initial fluorescence is weak, and it turns into bright yellow fluorescence after reacting with NO.

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