Tcs sp5 and sp8

To assess CM necrosis, 2-month-old C57BL/6J male mice were used. Mice received i.p. injection of 100 µg/100 µL of a monoclonal antibody against cardiac myosin (MF-20, ID: AB_2147781, DSHB) 6 h after LCC/saline administration. All animals were sacrificed 1 day after cardiac myosin administration and the heart was fixed with 4% paraformaldehyde (PFA). The number of necrotic MF-20 positive CMs was manually counted in cardiac cross-sections for each power field using a 63X objective for a total of 20 fields and the number of MF-20 positive CMs was expressed as a percent fraction of the total CM number per mm2. All stainings were acquired and analyzed using confocal microscopy (LEICA TCS SP5 and SP8).

Mouse brains were fixed in 4% paraformaldehyde in PBS at 4 °C for overnight, embedded in paraffin and sectioned at 7 μm in the coronal or sagittal planes. For histological analyses, sections were rehydrated and stained with hematoxylin and eosin. For immunostaining, after rehydration, sections were incubated in boiling citrate buffer (10 mM, pH6.0) for 30 min for antigen retrieval. After three washes with PBS, primary antibodies (Supplementary Methods) were incubated with 5% normal goat or donkey serum in PBS at 4 °C for overnight. Sections were then washed in PBS and incubated with the secondary antibodies. Primary antibodies information can be found in Supplementary Information. For secondary antibodies, Alexa Fluor 488-conjugated goat anti-mouse, anti-rabbit or anti-chicken, 647 conjugated goat anti-mouse (Invitrogen), Cy3-conjugated goat anti-mouse or anti-rabbit (Jackson ImmunoResearch), and biotin-conjugated goat anti-rabbit (Vector Labs) were used. Images were acquired using Axioplan 2 (Carl Zeiss) and confocal microscopes (TCS SP5 and SP8, Leica) and analysed with LAS AF (Leica) and Photoshop (Adobe).