Affinipure goat anti mouse secondary antibodies

Anchor oligonucleotides (Supplementary Table 1) were conjugated to secondary antibodies for immunolabeling of cardiac samples. Lyophilized oligonucleotides were resuspended in PBS (pH 7.4) to 100 µM and kept at −20 °C for long term storage until required for conjugation. AffiniPure Goat Anti-Mouse secondary antibodies (affinity purified, #115-005-003, Jackson ImmunoResearch, PA) were conjugated using click-chemistry as described by Schnitzbauer et al.^{2 (link)} Briefly, the antibody was incubated with 10-fold molar excess DBCO-sulfo-NHS-ester (Jenabioscience, Germany) for 45 min. The reaction was quenched with 80 mM Tris-HCl (pH 8.0) for 10 min and then desalted using 7 K MWCO Zeba desalting columns (Thermo Fisher). A 10-fold molar excess of the azide modified oligonucleotide was then incubated with the DBCO-antibody mixture overnight at 4 °C. Subsequently the antibody was purified using 100 K Amicon spin columns (Sigma). The absorbance of the oligonucleotide-conjugated fluorophores (Cy3 or Cy5) was recorded with a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham) and used to quantify the degree of labeling for each conjugation, typically achieving >1–3 oligonucleotides per antibody.