Anti mac 2

Paraffin-embedded tissue sections of 4 μm thicknesses were deparaffinized with xylene, dehydrated in gradually diminishing concentrations of ethanol, and treated with 3% hydrogen peroxidase in methanol for 10 min to block endogenous peroxidase activity. The tissue sections were immersed in a 10 mM sodium citrate buffer (pH 6.0) for 5 min at 95 °C. The last step was repeated using a 10 mM sodium citrate solution (pH 6.0). The sections were allowed to stay in the same solution while cooling for 20 min, following which they were rinsed in PBS. Then, the sections were incubated with a primary antibody (1:100 dilution) for 1 h at 37 °C. The primary antibody was as follows: anti-neutrophil gelatinase-associated lipocalin (NGAL, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-kidney injury molecule-1 (Kim-1, formerly called Tim-1, Abcam, Cambridge, MA, USA), anti-Mac-2 (formerly called Galectin-3, Abcam), and anti-CD4 (Abcam). The signal was visualized using an Envision System (DAKO, Carpinteria, CA, USA) for 30 min at 37 °C. 3,3′-Diaminobenzidine tetrahydrochloride (DAB) was used as the coloring reagent, and hematoxylin was used as the counter-stain. The slides were examined using a slide scanner (Pannoramic MIDI) and analyzed with iSolution DT software (IMTechnology, Vancouver, BC, Canada).

Atrial samples were fixed in 4% paraformaldehyde, embedded in paraffin, and then sectioned (5 μm). The staining of Hematoxylin and eosin (H&E) and Masson’s Trichrome were performed on the atrial sections in accordance with the standard procedure (Li et al., 2018 (link), 2019 (link); Zhang et al., 2020 (link)). The immunohistochemistry staining was performed with anti-α-smooth muscle actin (α-SMA) (1:200, Abcam, MA, United States) and anti-Mac-2 (1:200, Abcam) as described (Wang et al., 2018 (link)). Cryosections were stained with the dihydroethidine (DHE, 1 μM in PBS) for 30 min at 37°C. Fluorescence was detected using Nikon Labophot 2 microscope (Nikon, Tokyo, Japan).

Immunological staining of brain sections was performed using a floating method, as previously described44 (link). Primary antibodies were diluted as follows; 1:500 for anti-ionized calcium binding adaptor molecule-1 (Iba-1) (Wako Pure Chemicals, Osaka, Japan), 1:250 for anti-Mac-2 (Abcam, Cambridge, UK), and 1:500 for anti-myeloperoxidase-1 (MPO-1) (Abcam, Cambridge, UK). Experiments were repeated at least three times and representative images are presented.