Af114

Mouse heart tissue samples were fixed in 10% formalin for at least 48 hours and embedded in paraffin. Cardiac tissues were cross-sectioned into 4-5 μm-thick slides. Masson Trichrome staining (Sigma, HT15-1KT) and TUNEL staining (Roche, 12156792910) were performed following the manufacturer's instruction and previously described in detail 24 , 26 (link). For the identification of endothelial cells and pan immune cells, CD31 (R&D, AF3628), CD45 staining (R&D, AF114). Cardiomyocytes were identified by staining for α-Sarcomeric Actin (α-SARC, Sigma-Aldrich, A2172). Images were acquired using the Niko Eclipse Ti fluorescence microscope using 4X, 10X, 20X and 40X objectives and planimetry analysis using ImageJ.

Mouse heart tissue samples were fixed in 10% formalin for at least 48 hours and embedded in paraffin. Cardiac tissues were cross-sectioned into 4–5 μm-thick slides. Masson Trichrome staining (Sigma Aldrich, USA) was performed following the manufacturer’s instructions and previously described in detail. For the identification of endothelial cells and pan-immune cells, CD31 (AF3628; 1:30 dilution, R&D Systems, USA), CD45 staining (AF114; 1:50 dilution, R&D Systems, USA), and CD206 (AF2535; 1:50 dilution, R&D Systems, USA;) were used, respectively. Donkey anti-goat (A-21432, ThermoFisher, USA) or donkey anti-rabbit (A-31572, ThermoFisher, USA) secondary antibodies were used at a dilution of 1:100. Images were acquired using the Eclipse Ti fluorescence microscope (Nikon, Japan) using 20x objective and planimetry analysis using ImageJ.

For hair cell and macrophages counting, mice were sacrificed at P18. The cochleae were perfused with 4% paraformaldehyde and decalcification with 10% sodium EDTA solution for 48 h, each stretched cochlear preparation was isolated from the cochlea. Cochlear explants in each group were fixed with 4% paraformaldehyde for 1 h at room temperature. After being rinsed three times with 0.1% Tween-20 in PBS (PBST), the flattened cochlear preparations or cochlear explants were incubated in a blocking solution of 5% Bovine Serum Albumin, followed by incubation with primary antibodies: polyclonal rabbit anti-myosin7a antibodies (25-6790, Proteus Bio-Sciences), polyclonal goat anti-sox2 antibodies (sc-17320, Santa Cruz Biotechnology), polyclonal rabbit anti-Cx26 antibodies (512800, Invitrogen) and goat anti-CD45 polyclonal antibody (AF114, R&D Systems) diluted in PBS overnight at 4 °C. The samples were washed three times with PBST and then incubated with secondary fluorescent antibodies (1:200 dilution, Antgene, China) for 2 h at room temperature. Nuclei and F-actin staining were labeled with DAPI and phalloidin (P5282; Sigma, USA) for 10 min. The samples were visualized under a laser scanning confocal microscope (Nikon, Tokyo, Japan).

Murine melanoma tumors were resected and paraffin-embedded. Tissue sections of 5 µm thickness were prepared using a cryostat (Leica, Buffalo Grove, IL, USA). Tissues were deparaffinized and rehydrated using serial EtOH dilutions. Tissue sections were incubated in H₂O₂ for 30 min at room temperature. Sections were stained with hematoxylin and counterstained with eosin. For IHC, rehydrated slides were incubated in blocking buffer (DAKO non-serum protein block, Agilent, Santa Clara, CA, USA) for an hour followed by primary CD45 antibody (1:1000 dilution, AF114, R&D Systems) incubation overnight. Sections were stained with horseradish peroxidase-labeled secondary antibody (1:200 dilution, BA-9500, Vector Laboratories, Burlingame, CA, USA) for 30 min followed by DAB application (DAKO, Agilent). Slides were counterstained with hematoxylin. Slides were washed three times with PBS between each stain. Slides were imaged using the Olympus DP80 (Center Valley, PA, USA) imaging system.

Whole mount tissues or cryosections were blocked with 5% donkey serum, 0.1% Triton-X 100, 1% BSA, and 0.02% sodium azide (NaN3) in PBS at pH 7.4 for 1 h at room temperature (RT). Samples were then incubated in primary antibodies overnight at 4 °C. The following primary antibodies were employed: rabbit anti-myosin VIIa (1:200; 25-6790; Proteus BioSciences), goat anti-CD45 (1:100; AF114, R&D systems., Minneapolis, MN, USA), rat anti-F4/80 (1:150, ab6640, Abcam Inc., Cambridge, MA, USA), rat anti-Ly-6G/C (1:100: ab2557, Abcam Inc, USA). The specimens were incubated with secondary antibodies diluted in 0.1% Triton-X 100, 0.1% BSA and 0.02% NaN3 in PBS for 1 h at RT. The secondary antibodies were conjugated with Alexa Fluor 488, 546, and 647 (1:500; A11055, A10040, and A31571, Life Technologies, Carlsbad, CA). After washing with PBS, specimens were mounted in ProLong® Gold Antifade Reagent with DAPI (Cell signaling, #8961 Danvers, MA 01923) and placed under a cover slip. Images were captured using a LSM700 confocal microscope (Zeiss, Germany) at 10X magnification.