Mounting medium

Cells were fixed with 4% formaldehyde (Wako, Osaka, Japan), permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin prepared in phosphate-buffered saline (PBS). Cells were incubated with an anti-human E-cadherin (ab40772, Abcam) or vimentin antibody (ab92547, Abcam) at 4°C overnight and then with donkey anti-rabbit IgG (ab150073, Alexa Fluor® 488, abm Inc.) or goat anti-rabbit IgG (# A-11011, Alexa Fluor® 568, Thermo Fisher) for 2 h. Finally, cells were washed and mounted with mounting medium containing DAPI (Abcam). Images were acquired using a confocal microscope (LSM800; Carl Zeiss, Inc., Jena, Germany).

Tissue sections were deparaffinized and then blocked with CAS‐Block (Invitrogen, CA) for 1 h. Incubated the sections with primary antibody (NOX4, 4HNE, CD31; Abcam, USA) for 2h at 37°C followed by incubation with secondary antibody linked with HRP for 1 h at 37°C. Place sections in solution for DAB reaction for 3min. At last, sections were then stained with haematoxylin for 2 min, washed with PBS, and mounted with mounting medium (Abcam, USA). Capillary density was assessed based on the capillary/myocyte nucleus (C/M) values as previously described.²⁵

Cells were fixed using 4% paraformaldehyde in cold DPBS and permeabilized using 10.5% Triton X-100 in DPBS for three minutes. A blocking solution with 5% bovine serum albumin (BSA) was then added. The cells were incubated with IL-6, FLG, IL-4, and IL-1β primary antibodies overnight, followed by incubation with a FITC-labeled secondary goat anti-Rabbit IgG (H&L) antibody (Abcam) for one hour. Nuclei were stained with 4′,6-Diamidino-2-phenylindole, dihydrochloride (DAPI) included in the mounting medium (Abcam). After, all cells were visualized using a Leica SP8 confocal microscopy (Philadelphia. PA, USA).

At first, use the cold PBS to wash cultured cardiomyocytes for three times. Then, 4% paraformaldehyde was utilized to fix cells for 15 min. Cardiomyocytes were then washed with cold PBS again so that the cardiomyocytes could be blocked with 10% normal goat serum and 1% BSA (Sigma, USA). Next, cardiomyocytes were incubated with anti-α-actinin (1:500, Sigma, USA) in a dark room at 4 °C overnight and were further incubated with secondary antibody (1:1000; Molecular Probes, USA) at a normal temperature for 1 hour. Next, the slides were covered with coverslips. The nucleus was stained with mounting medium (Abcam, USA) which containing Hoechst33342 (Beyotime, China). At length, a fluorescence microscope (Olympus, Japan) helped us to observe and photograph cells. Cell surface area was quantified by detecting 50 random cells from three independent experiments. Thereafter, the average value was used for further analysis. Cell surface areas were assessed by ImageJ software by observing the 100–200 cardiomyocytes in 30–40 fields.

The specimen sections were deparaffinized and rehydrated. Then, endogenous peroxidase was blocked using a hydrogen peroxide block (Abcam, UK). The specimens were washed, and antigen retrieval was performed by heating in a 10 mM citrate buffer. After protein block solution (Abcam, UK) was applied to reduce nonspecific background staining, the specimens were incubated with primary antibodies overnight at room temperature in a humidified chamber. The antigen-antibody complex was then detected using a Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC Kit (Abcam, UK), following the manufacturer's instructions. The sections were counterstained with hematoxylin solution (Mayer's modified, Abcam, UK) and dehydrated before mounting with mounting medium (Abcam, UK). As a negative control, the primary antibody was replaced with SignalStain antibody diluent (Cell Signaling Technology, USA). The stained specimens were examined under a Motic BA210 microscope.

Spleens and draining lymph nodes (dLNs) were isolated, spleens were split to three pieces, tissues were embedded in Tissue-Tek® (Sakura, Staufen) and frozen at −80 °C. 8-µm thick slices were cut with Cryotom (Thermo Scientific). Slices were fixed with acetone, air dried, and frozen at −20 °C. For stainings, slides were thawed at RT for 15 min, rehydrated in PBS or PBS 1% BSA (dLNs) for 5 min, blocked for 30 min in IHC-Blocking buffer or PBS 1% BSA (dLNs) and then stained with the following antibodies: CD4-Alexa Fluor 647, B220-Alexa Fluor 488, IgM-Cy5 (Southern Biotech), IgD-Alexa Fluor 488, MoMa-Biotin (Southern Biotech), streptavidin-TRITC (Southern Biotech), and DAPI (Abcam) in IHC-staining buffer for 30 min at RT in dark humid chamber. Then, slides were washed twice for 10 min with 0.05 % Tween20 in PBS and one time with PBS only. Slides were then mounted with mounting medium (Abcam) and analyzed with Zeiss Axio Lab.A1 (Carl Zeiss) or Leica SP5 Confocal microscope. B cell follicles were identified by B220 + areas at ×100 total magnification and borders drawn by isolate/draw tool. Then, a channel was switched to CD4 signal, and CD4 MFI of T cells within the isolated region analyzed by Fiji^{70 (link)}. In average about 3–6 non-sequential follicles were counted per animal. Blind analysis was applied to all image analysis.