B7264

The recombinant human ASM (2 µg ml⁻¹, Genscript) in ELISA coating buffer (Abcam, ab210899) was coated onto MaxiSorp ELISA plates (Thermo Fisher Scientific) overnight at 4 °C. Wells were incubated with blocking buffer (Abcam, ab210904) for 2 h at RT, washed with wash buffer (Abcam, ab206977). ASM antibody (23A12C23) or diluted plasma in blocking buffer were incubated for 2 h at RT. Following washing with wash buffer, Anti-mouse IgG/Biotin (Sigma-Aldrich, B7264) in blocking buffer was added and incubated for 1 h at RT. After standard washing, wells were further incubated with streptavidin HRP (Abcam, ab210901) in blocking buffer for 1 h at RT and results were developed with TMB substrate (Abcam, ab210902). Absorbance was measured at 450 nm using a Varioskan LUX Multimode Microplate Reader (Thermo Scientific). The K_D was analyzed using the SigmaPlot 13.0 software. Each experiment was performed in triplicate. The ASM titers were defined as the dilution factor referring to 50 % of the maximal optical density (ODmax/2).

Adult hearts with pericardial nephrocytes from 7-day-old females were dissected in artificial Drosophila haemolymph according to Selma-Soriano et al. (2018a) (link), fixed with 4% paraformaldehyde (PFA) in PBS for 20 min and permeabilized by washing three times with PBS containing 0.3% Triton X-100 (PBS-T) for 10 min. Then, hearts were blocked in PBS-T containing 0.5% bovine serum albumin for 30 min at room temperature and incubated with the corresponding primary antibody (1:100) overnight at 4°C. Primary antibodies used were anti-Rph (Abcam, ab3338; with 50% of homology), anti-Hrs (DSHB, AB 2722114), anti-Rab3 and anti-Rab27 (BD Bioscience, 610379 and 558532, respectively) and anti-Klf15 (Ivy et al., 2015 (link)) (Abcam, ab22851; with 59% of homology). After three washes with PBS-T, the secondary antibodies (1:200), AlexaFluor-647 donkey anti-rabbit (Life Technologies, A31573), anti-mouse biotinylated (Sigma-Aldrich, B7264) and anti-rabbit biotinylated (Sigma-Aldrich, B8895) were incubated for 2 h at room temperature. Hearts with pericardial nephrocytes were then incubated with ABC solution (ABC kit, VECTASTAIN) for 30 min at room temperature, followed by washes and incubation with streptavidin-Texas Red (Vector Laboratories, 1:500). All images were taken using an LSM 800 confocal microscope (Zeiss) and were processed using ZEN software.

AGE-modified LDL were used for coating (200 μg/mL of each in PBS pH 7.4) microtiter plates (Nunc MaxiSorp, Denmark) in an overnight incubation at 4°C. Coated plates were washed with PBS with 0.05% Tween-20 and thereafter blocked with SuperBlock in Tris-buffered saline for 5 minutes at room temperature followed by an incubation of mouse serum diluted 1:50 in TBS-0.05% Tween-20 for 2 hours at room temperature and overnight at 4°C. After washing, bound antibodies were detected by using biotinylated goat anti-mouse IgM (AP500B, Millipore, USA) or IgG (B7264, Sigma, USA) or rat anti-mouse IgG1 or IgG2a secondary antibodies (RMG115 and RMG2a15, Millipore, USA) that were incubated for 2 hours at room temperature. The plates were washed, and bound biotinylated antibodies were detected by alkaline phosphatase–conjugated streptavidin (V559C, Promega, USA). The color reaction was developed using phosphatase substrate kit (Pierce). Absorbance at 405 nm was measured after 1 hour incubation at room temperature. Mean values were calculated after subtraction of background absorbance (n = 4 per mouse).