D8200

Sections of OCT-embedded kidneys were 4 μm in thickness and subsequently inoculated at room temperature utilizing 5% horse serum for 60 min for blocking the non-specific binding sites. The slides were next inoculated overnight in a humid chamber under a temperature of 4 °C after dilution 1:200 in PBS with primary antibody Pan-Kcr, H3K9cr, α-SMA, COL6, γH2AX. The equivalent secondary antibody (1:500 dilution, 111-025-003, Jackson ImmunoResearch, West Grove, PA, USA) was utilized for 60 min. Slides were re-cleaned, stained with DAPI (dilution 1:500, D8200, Solarbio, Beijing, China), and sealed through coverslips. For cells, formaldehyde was used for fixation for 15 min, and the primary antibody γH2AX was added to the slides at 4 °C overnight. Secondary antibodies (1:500 dilution, 111-545-144, Jackson ImmunoResearch, West Grove, PA, USA) was applied for 60 min at room temperature. After washing, the cells were stained with DAPI (dilution 1:500, D8200, Solarbio, Beijing, China). The co-staining of IL-1β with H3K9cr, IL-1β with iNOS and F4/80 were performed by Opal reagent (Y6084S, Y6088S, Y6094S, Uelandy, Suzhou, China) according to the guidelines of the manufacturer. Images were gathered under an AxioCamHRc digital camera (Carl Zeiss, Jena, Germany) via utilizing the ZEN 2012 microscopy software (blue version).

OCT-embedded kidney sections (4 μm) were incubated with phosphate-buffered saline (PBS) containing 5% horse serum for 1 h at room temperature to block non-specific binding sites. Then the specimens were incubated with primary antibody anti-FABP4 (1:200, 12802-1-AP, Proteintech Group, Chicago, USA) in a humidified chamber overnight at 4°C. After washing, the secondary antibody (1:500, 111-025-003, Jackson ImmunoResearch, West Grove, PA, USA) was used for 1 h. Fluorescein-labeled Lotus tetragonolobus lectin (LTL) (1:400, FL-1321, Vector Laboratories, CA, USA) was applied for identifying proximal tubules. The samples were washed again and then stained with DAPI (1:500, D8200, Solarbio, Beijing, China) and finally sealed with coverslips. Images were acquired by an AxioCamHRc digital camera (Carl Zeiss, Jena, Germany) at ×200 magnification with ZEN 2012 microscopy software (blue edition).

Each heart was incised transversely along the lower edge of the atrium, embedded with paraffin and cut into 5-µm-thick slices. The sections were dewaxed, rehydrated and blocked with 0.1% (v/v) Triton X-100/0.25% bovine serum albumin (BSA), and were subsequently incubated with various primary antibodies, fluorescently-labeled secondary antibodies and 4, 6-diamidino-2-phenylindole (DAPI, Solarbio, D8200). The following primary antibodies were used: rabbit anti-cardiac troponin T (CTNT, Abcam, ab209813, 1:500), and rabbit anti-CD68 (Boster, BA3638, 1:200). CY3-conjugated anti-rabbit secondary antibody (Abcam, ab6939, 1:200, anti-CD68), or CY5-conjugated anti-rabbit secondary antibody (Abcam, ab6564, 1:500, anti-CTNT) was applied following primary antibody incubation. The images were observed under a fluorescence microscope (Nikon, ECLIPSE 80I). Six 40× fields were randomly selected and taken photos to count the average number of positive cells.