Alexa fluor 594 conjugate

The transfected cells were fixed in 4% paraformaldehyde lasting 30 min. And, 0.5% Triton X-100 was applied to cover the slides and incubated at room temperature for 10 min. The sample was blocked with 10% goat serum for 1 h, and then washed thrice with 1% serum blocking solution. The primary antibody against MCU (1:100; sc-515,930; Santa Cruz, USA), MICU1 (1:1000; ab190114; Abcam, USA) and MICU2 (1:1000; ab101465; Abcam, USA) was added dropwise to the slices. After washing six times with 1% serum blocking solution, the sections were incubated with secondary antibodies Alexa Fluor® 488 Conjugate (1:100; ZF-0512; ZSGB-BIO, China) and Alexa Fluor® 594 Conjugate (1:100; ZF-0513; ZSGB-BIO, China) at room temperature for 2 h. Then, the sections were incubated by DAPI at room temperature for 5 min in the dark. The images were captured using an immunofluorescence microscope. The Image-Pro Plus 6.0 image processing system was used to measure the average optical density of the protein.

Following deparaffinization and hydration, specimens were cut into 4 μm thickness. The sections were incubated with fluorescent substance-labeled primary antibodies against Keap1 (1 : 200; Proteintech; China; 60027-1-Ig), Nrf2 (1 : 100; Proteintech; China; 16396-1-AP), and HO-1 (1 : 100; Proteintech; China; 27282-1-AP) overnight at 4°C. Then, sections were incubated with secondary antibodies including Alexa Fluor® 488 Conjugate (1 : 100; #ZF-0512, ZSGB-BIO, China) and Alexa Fluor® 594 Conjugate (1 : 100, #ZF-0513, ZSGB-BIO, China) at room temperature for 2 h. The images were observed under a confocal microscope (Olympus, Japan).