Innoscan 300 scanner

We measured the relative concentrations of a total of 170 proteins (see Additional file 1, Supplementary Table S2) with antibody arrays (RayBiotech Inc., Peachtree Corners, GA, USA) according to the manufacturer’s instructions. Briefly, a custom glass-based antibody array targeting the 170 proteins of interest was built, 100 μl of diluted serum sample was added to each well, incubated overnight at 4°C, and then extensively washed. We then incubated the wells with biotin-conjugated antibodies specific to the different proteins. Membranes were developed with Alexa Fluor^® 555-conjugated streptavidin (Thermo Fisher Scientific, Waltham, MA, USA). The slides were scanned with 532 nm excitation and extracted using an InnoScan^® 300 Scanner (Innopsys, Carbonne, France).

Total protein was isolated from the mouse heart using RIPA (Applygen) and the supernatant was analyzed by a glass-based and sandwich-based antibody microarray to measure 20 cytokines quantitatively (RayBiotech). Each cytokine was analyzed in quadruplicate per array. Then, 100 µl supernatant were loaded to each well, incubated overnight at 4 °C and then extensively washed. A biotin-labeled detection antibody was incubated for 2 h followed by the application of AlexaFluor 555-conjugated streptavidin at room temperature for 1 h. The slides were analyzed by an InnoScan 300 Scanner (Innopsys) with 532 nm excitation and 635 nm emission. We obtained raw data (images) from the scanner and integrated spot intensities (tab-delimited .txt file) using Mapix v.7.3.1 software. Data visualization was obtained by Q-Analyzer Software (RayBiotech). All raw data were log₂-converted for statistical analysis.

The concentrations of 200 serum proteins were measured simultaneously using the Human Cytokine Array Q4000 (QAH-CAA-4000, RayBiotech, Georgia, USA) as previously described (11 (link)). Briefly, after incubated with blocking buffer for 60 min at room temperature (RT), the arrays were added with 100 µL of 2-fold diluted serum and were incubated at 4°C overnight. On day 2, the arrays were added biotin-labeled detection antibody for 1 hour (h) at RT followed by extensive washing. Alexa Fluor 555-conjugated streptavidin was added and incubated for 1 h at RT. The signals were scanned, and data were extracted by an InnoScan 300 scanner (Innopsys, Carbonne, France).