Alexa fluor 488 conjugated rabbit igg

Identification of NETs in this study was similar to a previously published protocol.^{10 (link)} Briefly, 2 × 10⁵ BMDNs were seeded onto poly-l-lysine-coated coverslips (Sigma-Aldrich). In experiments without stimulation or with stimulation with 2% serum (extracted from pristane-treated WT or Mfge8^−/− mice for 6 months), the cells were incubated for 2 h. In experiments with 1 μg/ml LPS, 100 nM PMA or 100 ng/ml MIP-2 stimulation, the incubation was for 4 h. NETs were stained with rabbit anti-histone H3 (citrulline R2+R8+R17) antibody and mouse anti-MPO antibody, followed by incubation with Alexa Fluor 647-conjugated mouse IgG and Alexa Fluor 488-conjugated rabbit IgG (Abcam). DNA was stained with DAPI. Images were collected with Olympus BX51 microscope and Qimaging camera, typically at original × 400 magnification.

H9c2 cardiac cells were grown on sterilized coverslips. After treatment with homocysteine alone, or in combination with Lu AF58027 or TUDCA, the cells were fixed with 4% paraformaldehyde (15 min), permeabilized with 0.1% Tween-20 for 5 min and then blocked with 2% BSA in PBS for 1h. The cells were then incubated overnight at 4°C with primary antibody targeting α smooth muscle actin (1:500; Abcam, Cambridge, UK), followed by incubation with Alexa Fluor 488-conjugated Rabbit IgG (1:1000; Abcam) for 1 h at room temperature. Nuclear DNA was labeled with DAPI. Images were captured with a fluorescence microscope (Olympus DP73, Tokyo, Japan).