Lsm 880 laser confocal fluorescence microscope

Cells were fixed with 4% paraformaldehyde at room temperature for 15 min. Then, they were permeabilized with 0.1% Triton X-100 for 20 min. Subsequently, the fixed cells were washed with PBS and blocked with 5% goat serum at room temperature for 30 min. The corresponding fluorescent secondary antibodies were incubated at room temperature for 2 h. After washing with PBS, the cells were sealed using DAPI reagent (Beyotime, Shanghai, China) and imaged using a Zeiss LSM 880 laser confocal fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Paraffin sections of lung tissue underwent a deparaffinization and rehydration processes before being subjected to antigen retrieval in heated sodium citrate buffer (10 mM) maintained at a temperature of 95 °C for 30 min. The follow-up procedure was the same as for cellular immunofluorescence.

To observe Nrf2 localization of MLE-12 cells. Cells were then fixed in 4% formaldehyde and permeabilized with 0.1%Triton X. The cells were probed with Nrf2 antibodies followed by Alexa Fluor 488-conjugated secondary antibodies (Thermo Fisher Scientific, USA). To visualize the nuclei, cells were then treated with 1 μg/ml DAPI for 10 min and then washed by PBS. Finally, added the anti-fade mounting medium and collected images using Zeiss LSM 880 laser confocal fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
To observe the expression of Nrf2 in mouse lung tissue. Paraffin slides were rehydrated in alcohol with decreasing concentrations and then placed in 10 mM sodium citrate buffer heated to 95 °C for 30 min for antigen retrieval. Then, the slides were permeabilized with 0.5% Triton-X and blocked with 5% BSA for 2 h. The slides were probed with Nrf2 antibodies followed by Alexa Fluor 555-conjugated secondary antibodies (Thermo Fisher Scientific, USA). The slides were incubated in DAPI for 10 min and then washed. Finally, the slides were mounted with an anti-fade mounting medium and collected images using Zeiss Axio Imager 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany).