Alexa fluor 647 goat anti mouse igg1

Biopsies of 5mm diameter were taken with a punch needle from the MI border zone or matching regions from control hearts. After immersion in 30% sucrose solution, slices of 80μm thickness were obtained by cryosectioning in a depth of 0.4 to 1.6mm below the epicardium as previously described (Seidel et al., 2016 (link)). Immediately after sectioning, and after each incubation step during staining, slices were rinsed in PBS.
The slices were labelled for sarcomeric actinin by incubation in anti-alpha-actinin antibody (ab9465, Abcam, Cambridge, UK) at a dilution of 1:200 in blocking solution (5% bovine serum albumin, 5% goat serum and 0.25% Triton X-100 in PBS) for a minimum of 12 h. Goat anti-mouse IgG1 Alexa Fluor 647 (A-21240, Thermo Fisher Scientific, Waltham, MA, USA) was then applied at 1:200 dilution together with 300nM 4′,6-Diamidino-2-Phenylindole, dilactate (DAPI, D3571, Thermo Fisher Scientific, Waltham, MA, USA) in blocking solution for a minimum of 12 h. Finally, we applied CF488A-conjugated wheat germ agglutinin (WGA, Biotium, Hayward, CA, USA) at 40 μg/mL in PBS for a minimum of 4h.
Tissue slices were mounted on glass slides using Fluoromount-G (#17984-25, Electron Microscopy Science, Hatfield, PA, USA) and covered with coverslips of thickness 1.5. Samples were left to dry at room temperature for at least 1d before imaging.

For immunohistochemical analysis, U2OS cells were cultured on 96-well optically clear plates (Greiner Bio-One), transfected as previously described, then fixed in 4% PFA (Electron Microscopy Sciences) diluted in PBS (Gibco) and washed with 0.3M glycine (Sigma) in PBS to quench PFA. Samples were blocked and permeabilized in a PBS solution containing 8% donkey serum (Jackson ImmunoResearch), 8% goat serum (Cell Signaling Technologies), and 0.3% Triton-X 100 (Sigma) for one hour, followed by primary antibody incubation in 1% BSA (Fisher Bioreagents), 1% goat serum, and 0.25% Triton-X overnight at 4°C. Samples were washed 3 times with PBS containing 0.1% BSA and 0.1% Triton-X 100 before incubating with fluorophore-conjugated secondary antibodies in PBS with 0.05% Triton-X 100 and 1% BSA at room temperature for one hour. Cells were washed with PBS with 0.1% Triton-X, stained with DAPI, and then covered with Mounting Media (Ibidi) before imaging. Primary antibody, HA-Tag 6E2 (Cell Signaling, 2367), was used at a 1:100 dilution as per manufacturer’s instructions. Secondary antibodies used were goat anti-mouse IgG1-Alexa-Fluor 647 (Thermo Fisher, A21240) and Anti-Mouse IgG1 CF 633 (Sigma, SAB4600335). Confocal images were taken using a Zeiss Airyscan LSM 880 followed by image processing in Zen 2.3 (Zeiss).