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.