Lycopersicon esculentum lectin lel

Cells were lysed in 1% triton lysis buffer. For untagged LRP6 protein, IP was performed with mouse monoclonal anti-LRP6 (Abcam, Cambridge, UK) and protein G agarose (Thermo Fisher Scientific, Idstein, Germany). For flag-tagged LRP6 protein, IP was performed using anti-Flag M2 affinity gel (Sigma-Aldrich, Taufkirchen, Germany). IP products were used for lectin blotting. The PVDF membrane (Bio-Rad Laboratories, Feldkirchen, Germany) was blocked with RIPA buffer (50 mM Tris-HCL, 0.1% Trito X-100, 0.1% Sodium Deoxycholate, 150 mM NaCl, 0.1% SDS, adjusted to pH 7.4) for 1 h at room temperature. Amounts of 2 μg/mL biotinylated Lycopersicon esculentum lectin (LEL, Vector Laboratories) or 5 μg/mL biotinylated Concanavalin A (ConA) (Vector Laboratories, Newark, CA, USA) were prepared freshly in RIPA buffer and incubated with the membrane at 4 °C overnight. After washing with RIPA buffer, the membrane was incubated with 1/2500 streptavidin-HRP (GE HealthCare, Braunschweig, Germany) for 2 h at room temperature. The membrane was washed with TBST (50 mM Tris, 150 mM NaCl, 0.05% Tween-20, adjusted to pH 8.0) buffer before subjecting to ECL detection.

Formalin-fixed heart were embedded in paraffin or frozen in liquid nitrogen and sectioned into 5–8μm slices. Viable tissue and collagen were visualized by incubation with a 0.1% solution of Fast Green and a 0.1% solution of Sirius Red (Sigma) in 1.2% picric acid (1h, RT). Blood vessels were detected by incubation with either lectin conjugated with FITC (Lycopersicon Esculentum Lectin LEL, Vector Laboratories) or CD31 (Abcam) antibody and FITC-conjugated secondary antibody. Macrophages were stained with appropriate antibodies (CD206 or iNOS; Abcam) and fluorochrome-conjugated secondary antibodies. Human cells in a mouse tissue were identified by human lamin A+C, Nuclear Envelope Marker (Abcam). IL -6 was stained with anti-IL-6 antibody (Abcam) and Texas Red-conjugated secondary antibody. Nuclei were counterstained by DAPI.