Alexa fluor 647 goat anti mouse

The following antibodies were used for immunofluorescence or flow cytometry assays: mouse anti‐human CD11c monoclonal antibody (cat# 14‐0116‐82, Thermo Fisher Scientific); rat anti‐mouse Lamp‐1 antibody (clone 1D4B, Developmental Studies Hybridoma Bank); mouse anti‐human Lamp‐1 antibody (clone H4A3, Developmental Studies Hybridoma Bank); mouse anti‐human GM130 antibody (cat# 610822, BD Biosciences); rabbit anti‐human calnexin antibody (cat# ab112995, abcam); rabbit anti‐bordetella pertussis toxin antibody (cat# ab188414, abcam); rabbit anti‐human TGN46 antibody (cat# ab50595, abcam); rabbit anti‐cathepsin D antibody (cat# 219361, Millipore Sigma); Cy3‐donkey anti‐rat IgG (cat# 712–165‐153, Jackson Immunoresearch); donkey anti‐rabbit Alexa Fluor 647 (cat# A31573, Invitrogen); donkey anti‐rabbit IgG Alexa Fluor 555 (cat# A31572, Life Technologies); goat anti‐mouse Alexa Fluor 488 (cat# 11029, Thermo Fisher Scientific); goat anti‐rabbit Alexa Fluor 647 (cat# A21245, Thermo Fisher Scientific); goat anti‐mouse Alexa Fluor 647 (cat# A21235, Thermo Fisher Scientific); donkey anti‐mouse Alexa Fluor 555 (cat# A31570, Thermo Fisher Scientific). The following antibodies were used during blocking and opsonization assays: rat anti‐mouse CD11b (blocking antibody, clone: M1/70, cat# AB‐467108, Thermo Fisher Scientific) and human IgG (opsonization, I8640 or I4506, Millipore Sigma).

After post-ablation imaging, cardiac disks were fixed in 4% PFA for 24 h. Samples for cryosection were treated with 30% sucrose for 24 h prior to OCT embedding and then sectioned at a thickness of 5-7 µm. Before staining, samples were incubated with 0.2% Triton-X-100 for an hour followed by another 2 h incubation with the blocking buffer containing 5% BSA, 0.1% Triton X-100, 2% goat serum, and 1% glycine. Cardiac troponin T (cTnT) was stained by the primary antibody mouse anti-cTnT (1:200 dilution, cat#MS-295-P1, ThermoFisher, Waltham, MA, USA) and the corresponding secondary antibody goat anti-mouse AlexaFluor647 (1:500 dilution, cat#A21236, ThermoFisher, Waltham, MA, USA). Cell nuclei were stained with DAPI. Sectioned samples were also stained for connexin 43 (CX43) using rabbit anti-Cx43 (1:100 dilution, cat#ab11370, Abcam, Cambridge, UK) and goat anti-rabbit AlexaFluor488 (1:500 dilution, cat#A11008, ThermoFisher, Waltham, MA, USA). Samples were examined using a DMi8 fluorescence microscope (Leica, Wetzlar, Germany). To determine the fiber length of cTnT proteins, five 20× images were obtained from each condition and analyzed by using the Tubeness plugin in Fiji (ImageJ; National Institutes of Health, Bethesda, MD, USA) to identify fiber structures first and then using the Anamorf plugin [60 (link)] in Fiji to measure fiber length.

Cells were first washed with PBS (Thermo Fisher) and fixed with 4% PFA (MS). Cells were stained with the following primary antibodies and concentrations, Desmin (ab6322; Abcam; 1:250), PAX7 (Pax7‐c; DSHB; 5 μg/mL), MYOD1 (sc‐760; Santa Cruz; 1:50), MHC‐Alexa Fluor 488 (53‐6503‐82 [MF‐20]; Thermo Fisher; 1:100), α‐actinin (A7811; Sigma; 1:500) and myogenin (sc‐576; Santa Cruz; 1:200). The following secondary antibodies were also used together with non‐conjugated primary antibodies, Goat‐anti‐mouse Alexa Fluor 488 (A11001; Thermo Fisher; 1:500), Goat‐anti‐rabbit Alexa Fluor 594 (A11012; Thermo Fisher; 1:500) and Goat‐anti‐mouse Alexa Fluor 647 (A21235; Thermo Fisher; 1:500). DAPI (d9542; Sigma) was used as a nuclear counter stain according to manufacturer’s recommendations. Stained cells were imaged with a Zeiss fluorescence microscope.