4 hne bs 6313r

For immunofluorescence staining, antigen retrieval was performed with proteinase K (20 μg.mL⁻¹) at 37 °C for 1 h on paraffin sections. Primary antibodies against the following proteins were used: DMP1 (DF8825, 1:100, Affinity), GPX4 (ab231174, 1:100, Abcam), TfR1 (13-6800, 1:100, Invitrogen), 4-HNE (bs-6313R, 1:200, Bioss), RANKL (AF0313, 1:100, Affinity), and RUNX2 (12556 S, 1:100, Cell Signaling Technology). An M.O.M. kit (Vector Lab, USA) was used for the anti-TfR1 antibody. Alexa Fluor-conjugated secondary antibodies (Invitrogen) were applied for fluorescent labeling. Images were captured with a confocal fluorescence microscope (Olympus FV3000, Tokyo, Japan).

Cisplatin (HY-17,394) was purchased from MCE (New Jersey, USA). The TUNEL ApoGreen Detection Kit (40307ES20), the 1st Strand cDNA Synthesis Kit (11141ES60) and Hieff qPCR SYBR Green Master Mix (11201ES08) were purchased from YEASEN Biotechnology (Shanghai, China). The following primary antibodies were used in this study: anti-Bax (380,709), anti-collagen I (501,352), anti-α-SMA (380,653), anti-NOX4 (380,874), anti-TGF-β1(346,599), anti-p-smad2 (R26361), anti-p-smad3 (380,775), anti-smad2 (200,790), anti-smad3 (R25743). anti-beta tubulin (380,628), and anti-GAPDH (380,626), which were purchased from Zen BioScience (Chengdu, China). Rabbit anti-PCNA polyclonal antibody (bs-2007R) was obtained from Bioss (Beijing, China). Masson (G1340) and Sirus red stain (G1472) were obtained from Solarbio (Beijing, China). 8-OHdG (bs-1278R), 4HNE (bs-6313R), and 3NIT (bs-8551R) were purchased from Bioss (Beijing, China). Endogenous peroxidase blocker was obtained from Zhongshan Jinqiao (Beijing, China). Orthofluorescence microscope was purchased from ZEISS (Germany).

The expression of β-actin (#4970, 1:1000; Cell Signaling Technology, MA, USA), 4-HNE (bs-6313R, 1:1000; Bioss, MA, USA), BAG3 (NBP2-27398, 1:2000; Novus, CO, USA), Bcl-2 (bs-0032R, 1:1000; Bioss, MA, USA), caspase 3 (bs-0081R, 1:1000; Bioss, MA, USA), p-eNOS (ser1177, 1:1000; Cell Signaling Technology, MA, USA) and PARP (#9532, 1:1000; Cell Signaling Technology, MA, USA) from the damaged heart infarct areas after ischemia/reperfusion injury were evaluated by Western blotting as described before [26 (link)]. The data were recorded and analyzed with a luminescent image analyzer (ImageQuant LAS 4000 mini; GE Healthcare, Little Chalfont, UK).