Ant030

Mice were euthanized in the same way as before and perfused with pre-cooled saline, followed by 4% paraformaldehyde. After fixation for 3 days, the brains were sliced into coronal sections of 40 μm, incubated with 5% bovine serum albumin (BSA) for 1 h, and then incubated for 24 h at 4 °C with anti-CD68 (MCA1957, AbD Serotec, United Kingdom) and anti-MPO (60130, STEMCELL Technologies, Canada), both diluted to 1:200. Sections were rinsed 3 times, incubated at room temperature for 2 h with Alexa Fluor 488-conjugated antibody (ANT023, Antgene, China) for CD68 and Alexa Fluor 594-conjugated antibody (ANT030, Antgene, China) for MPO, both diluted to 1:400; washed; and then stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were obtained with an Olympus laser confocal microscope. To determine the number of CD68- and MPO-reactive cells, three randomly chosen slides (0.20–0.70 mm rostral to the bregma) from each mouse were processed with ImageJ software (NIH) to count the number of cells and calculate the mean. All outcomes were assessed by two different blinded investigators.

The immunofluorescence assay analyses using the frozen renal biopsies from patients, the frozen kidney sections from rats and the cell climbing films were performed as previously described. The following primary antibodies were used in this study: Mfn2 (1:200; Abcam, United Kingdom); WT1 (NB110-60011, 1:50; Novus, GER); Synaptopodin (65194, 1:50; Progen, GER); TOM20 (sc-17764; 1:50; Santa Cruz, United States); Calreticulin (ab92516; 1:100; Abcam, United Kingdom); PERK (1:50; Santa Cruz, United States). The Alex Fluor 488/594 donkey anti-rabbit/Mouse IgG (HL) (ANT023, ANT024, ANT029, ANT030; 1:100; Antgene, Wuhan, China) were used as secondary antibodies. The nuclei were stained with an anti-fluorescence quencher containing DAPI (ANT063; Antgene, Wuhan, China). A confocal microscope (Olympus, Japan) was used to observe all of the microscopic images. The Pearson’s correlation coefficients (PCC) have been calculated in immunofluorescence assay of TOM20 and CRT, which with a value between 1 and −1, was used to quantitative colocalization between two proteins (1 means perfect correlation; −1 means completely excluded; 0 means no relationship) (Schober et al., 2018 (link)).