Lci 4

Tissue samples were digested as follows: 10 mg pieces of murine postischemic hearts were digested with Collagenase II (1 mg mL⁻¹; C6885, Sigma) and DNase I (50 µg mL⁻¹; D4263, Sigma) for 1 h. Porcine biceps femoris samples were sectioned into 50 mg pieces, digested first with 1 mg mL⁻¹ papain (P3375, Sigma) for 30 min, followed by 1 mg mL⁻¹ Collagenase/Dispase (Roche) for 30 min. Cell suspensions were pressed through a cell strainer (70 µm) and centrifuged at 300 × g. Red blood cells were lysed with an ammonium‐potassium buffer washed twice and stained for viability with trypan blue for murine and LIVE/DEAD (Thermo Fisher) for porcine cells. Murine cells were immediately stained with a specific antibody for CD31 (BM4086 , Origene) for 15 min, followed by secondary antibody staining (A48265, Thermo Fisher). Porcine cells were fixed with 4% w/v paraformaldehyde, permeabilized with Triton X100 and stained for EGFP (CAB4211, Thermo Fisher) and CD31 (LCI‐4, Bio‐Rad), followed by secondary antibody staining (A32731 and A48255, Thermo Fisher). Fluorescence measurements were carried out in an LSRFortessa (BD Biosciences).

Microscopy was performed with a Leica THUNDER Imager Tissue (Leica Microsystems,) equipped with a 63XxNA1.40 oil immersion objective. Optical zoom was utilized where applicable. For fluorescence excitation, SOLA light engine (Lumencor) was used. Acquisition was performed with 2048² pixels. Image processing was performed using Leica LAS X software, with utilization of built‐in image deconvolution (small volume computational clearing).
The following antibodies were used for immunofluorescence staining: EGFP (CAB4211, Thermo Fisher), murine CD31 (BM4086, Origene), porcine CD31 (LCI‐4, Bio‐Rad), CRISPR‐Cas9 (7A9‐3A3, Novus Biologicals), AAV9 intact capsid (ADK9, Origene). Highly crossadsorbed Alexa Fluor conjugated secondary antibodies were used for fluorescent labeling (A‐11034, A48265, A‐11029, A‐11032, Thermo Fisher). GFP/CD31 double positive cells in immunofluorescence images were counted from ten sections with ImageJ.