Ab53494

Dimethylation of lysine 4 on histone 3 (H3K4me2) of the smooth muscle myosin heavy chain 11 (Myh11) promoter was detected by in situ hybridization (ISH) and Proximity Ligation Assay (PLA) as previously published^{24 (link)}. Briefly, the 2 kb promoter of Myh11 was amplified by PCR, cloned into pCR2.1 vector for amplification (TOPO cloning kit, ThermoFisher Scientific) and biotin labeled probes were generated by Nick Translation (Roche) using biotin-14-ATP (ThermoFisher Scientific). Probes were denatured in hybridization buffer (2X SSC, 50% high grade formamide, 10% dextran sulfate, 1 μg mouse Cot-1 DNA) for 5 min at 80 °C. Abdominal aorta sections were incubated with 0.5% pepsin at 37 °C for 15 min, followed by incubation with hybridization buffer containing biotinylated probe for 5 min at 80 °C then followed by incubation for 16–24 h at 37 °C. Following hybridization, slides were washed in 2X SSC, 0.1% NP-40 buffer. Sections were blocked, incubated with mouse H3K4dime (5 μg/mL, Millipore Sigma, clone CMA303) and rabbit Biotin (5 μg/mL, Abcam #ab53494) antibodies overnight at 4 °C, followed by incubated with secondary antibodies containing PLA probe at 37 °C for 1 h. Ligation and amplification were performed (Duolink detection kit Orange 555 nm) and mounting medium with DAPI was used. Immunofluorescence was quantified with Image J.

Human and baboon brain sections were rehydrated in ice-cold binding buffer (50 nM Tris-HCl [pH 7.4], 10 nM MgCl₂, 0.1 mM EDTA, and 0.1% BSA) for 15 min and incubated 1 hr at room temperature in the presence of biotinylated LCN2 (25 pg/mL−1). After washing in harvesting buffer (50 mM Tris-HCl [pH 7.4]), samples were fixed in 4% PFA for 15 min, washed in PBS, and incubated with rabbit anti-biotin antibody (ab53494, Abcam, Cambridge, UK) overnight at 4°C. The signal was visualized, after incubation with anti-rabbit Alexa Fluor 488 (A21206, Life Technologies, Carlsbad, CA) followed by DAPI counterstaining. To test for assay specificity, the procedure described above was performed in the presence of hundred-fold excess of non-biotinylated LCN2 (2.5 ng/mL). The binding was quantitated using the ‘Cell Counter’ analysis in ImageJ, by counting total cells (DAPI-stained/blue) and LCN2-positive cells (LCN2-stained/green) and subsequently calculating the percent of LCN2-positive cells in the two conditions.