ERNT

Guideline
(The following is a recommended experimental protocol for guidance only, and adjustments are required based on your specific needs)^[1].

1. Preparation of Working Solution
1.1 Concentration recommendations: 10 μM; 15 μM.

2. Staining Procedures
2.1 Sample types:
2.1.1 Adherent cells (HepG2).
2.1.2 Liver tissue samples: Isolate the liver organ and rinse it with PBS (pH 7.4).
2.2 Incubation conditions:
2.2.1 Adherent cells: Incubate with 10 μM ERNT (with continuous irradiation for 0, 5, 10, 15 and 20 min).
2.2.2 Liver tissue samples: Incubate with 15 μM ERNT.
2.3 Washing procedures:
2.3.1 Liver tissue samples: Rinse three times with PBS (pH 7.4) before staining.

3. Control Group Setup
3.1 Set up untreated adherent cells as the control group.
3.2 Set up untreated liver-injured mice as the control group.
3.3 Set up normal liver tissue as the reference group.

4. Detection and Analysis
4.1 Instruments: Fluorescence imaging system; In vivo imaging system.
4.1.1 Adherent cells: Green channel: Ex = 488 nm, Em = 500-550 nm; Red channel: Ex = 488 nm, Em = 570-620 nm.
4.1.2 Liver tissue samples: Ex = 520 nm, Em = 620 nm; Ex = 570 nm, Em = 710 nm.
4.2 Result analysis:
4.2.1 Changes in fluorescence intensity:
4.2.1.1 Higher fluorescence intensity indicates lower endoplasmic reticulum (ER) polarity; lower fluorescence intensity indicates higher ER polarity.
4.2.1.2 Fluorescence intensity decreases gradually from normal liver tissue to fatty liver, liver fibrosis, liver cirrhosis and finally liver cancer.
4.2.1.3 Compared with the untreated control group, the fluorescence intensity of the drug-treated liver injury group increases.
4.2.1.4 The fluorescence intensity ratio of green channel to red channel (I_green/I_red) in normal cells is higher than that in ER stress-induced cells.
4.2.2 Fluorescence localization: Mainly localized to the endoplasmic reticulum.
4.2.3 Color changes: Emits green and red fluorescence in cells; emits yellow to red fluorescence in liver tissue.