Ab53444

At the end of the protocol, mice were deeply anaesthetized and were transcardially perfused with a 0.2M phosphate buffer (PB)/4% paraformaldehyde (PFA, Sigma-Aldrich). Brains were removed and placed in 30% sucrose for 48h and 30μm thick freezing microtome sections were realized. Slices were blocked with PBS, 0.5% Triton, 3% bovine serum albumin (BSA, Sigma-Aldrich) for 2h and then incubated overnight with anti-CD68 (1/800, Abcam, ab53444) and anti-CD206 (1/1000, Abcam, ab64693) antibodies in PBS, 0.5% Triton, 1% BSA at 4°C. Sections were then incubated with an Alexa-555-conjugated anti-rat (1/200, Invitrogen, A18744) or an Alexa-488-conjugated anti-rabbit (1/200, Invitrogen, 10424752) as secondary antibodies in PBS, 0.5% Triton, 1% BSA containing Hoechst 33342 (10μg/ml, Sigma-Aldrich).

The brains were removed after perfusion with the fixative 4% paraformaldehyde and then immersed in 30% sucrose in PBS. Serial sections were cut on a freezing microtome, blocked, and incubated with the following primary antibodies: goat anti‐CD31 (diluted 1:250, R&D, AF3628), rabbit anti‐Iba1 (diluted 1:500, Wako, 019‐19741), rat anti‐CD68 (diluted 1:100, Abcam, ab53444), rat anti‐C3 (diluted 1:100, Abcam, ab11862), mouse anti‐AQP4 (diluted 1:100, Abcam, ab9512), rabbit anti‐GFAP (diluted 1:500, Abcam, ab7260), and rabbit anti‐MMP9 (diluted 1:500, Abcam, ab38898). After washing, the sections were incubated with the appropriate Alexa Fluor 488‐ or Alexa Fluor 555‐conjugated secondary antibody (diluted 1:1000, Invitrogen) and counterstained with DAPI. Images were captured with a Zeiss microscope (Zeiss, LSM780).

Zeiss LSM980 confocal microscope (ZEISS) and Stereo Discovery.V8 were used to acquire fluorescence signals and tissue morphology. The images were processed by ZEN software (blue edition). Histology and immunohistochemical (IHC) analyses were performed with formaldehyde-fixed hearts that were routinely processed to generate frozen sections. Briefly, hearts were fixed in 4% paraformaldehyde overnight at 4 °C and transferred to ×PBS, followed by optimal cutting temperature compound (OTC) embedding. Haematoxylin and Eosin staining was performed for morphological analysis. WGA staining was used for cross-sectional area (CSA) measurements for at least 30 cells per section and three independent heart sections per group. Masson’s trichrome staining was used to measure fibrosis. IHC analyses of Anti-cardiac troponin T mouse mAb (1:200, Abcam, catalog no. ab8295, clone no. 1C11), Anti-alpha smooth muscle Actin rabbit mAb (1:200, Abcam, catalog no. ab124964, clone no. EPR5368), Anti-CD31 rabbit mAb (1:200, Abcam, catalog no. ab222783, clone no. EPR17260-263), Anti-CD68 rat mAb (1:200, Abcam, catalog no. ab53444, clone no. FA-11) and Anti-Vimentin rabbit mAb (1:200, Abcam, catalog no. ab92547, clone no. EPR3776) were performed to detect the cardiomyocyte CSA and cell co-localization, and DAPI was used to stain nuclei.

After all MRI studies, mice were sacrificed and hearts were extracted. Heart samples were thoroughly washed in saline solution and embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA) and cut into 5 μm thick short-axis slices. To evaluate the necrotic myocardium, immunohistochemistry was performed on frozen tissue sections using a primary antibody against myoglobin (ab77232, Abcam, Cambridge, MA, USA) [24 (link), 25 (link)]. To detect necrotic myocardium, a HistoMouse™-MAX kit (Invitrogen, Waltham, MA, USA) was used to immunostain the sections, and color was developed using the Fast DAB with metal enhancer tablet set (Sigma-Aldrich). To analyze macrophage distribution, heart sections were stained with a primary antibody against CD68 (ab53444, Abcam) and Alexa Fluor 488-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA). The sections were counterstained with DAPI to visualize the nuclei. We evaluated colocalization of MNPs and macrophages in inflamed myocardium by color-coding the confocal laser scanning microscopic images using red for RITC (MNP), green for Alexa Fluor488 (macrophages), and blue for DAPI (nuclei). Stained tissue sections were digitally scanned using a slide scanner (SCN400, Leica, Oberkochen, Germany).