Ab88082

Tissues were lysed in RIPA buffer containing a protease inhibitor cocktail (Beyotime Institute of Biotechnology, Haimen, China). Total protein isolated from the myocardium was separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in Tris-buffered saline with 0.1% Tween (TBST) containing 5% nonfat dry milk for 1 h at RT and then incubated in universal antibody diluent (New Cell & Molecular Biotech, Suzhou, China) using the appropriate primary antibody overnight at 4°C. The primary antibodies used in this experiment were specific to the following antigens: iNOS (1 : 1000; ab210823; Abcam, Cambridge, UK), Arg1 (1 : 1000; ab91279; Abcam, Cambridge, UK), C/EBPα (1 : 1000; 18311-1-AP; Proteintech, Chicago, USA), PU.1 (1 : 2000; ab88082; Abcam, Cambridge, UK), SOCS1 (1 : 1000; YT4362; Immunoway, Newark, USA), CD63 (1 : 1000; ab213090; Abcam, Cambridge, UK), HSP70 (1 : 1000; ab2787; Abcam, Cambridge, UK), and TSG101 (1 : 1000; ab125011; Abcam, Cambridge, UK). The membranes were incubated with HRP-conjugated secondary antibodies after washing in 5% TBST. Finally, the protein bands were detected using a gel chemiluminescence imaging analysis system and the Immobilon Western Chemiluminescent HRP Substrate reagent (Millipore, Billerica, USA) and then were analyzed using ImageJ.

After transfection for 24 h, primary rat microglial cells were fixed with 4% PFA for 10 min and immunostained with primary antibodies against PU.1 (ab88082, Abcam, Berlin, Germany, 1:100) or FlOT1 (ab41927, Abcam, 1:100) at 4 °C overnight. Secondary antibody Alexa Fluor 555 donkey anti-mouse (A31570, Thermo Fisher Scientific) or Alexa Fluor 555 donkey anti-rabbit (A31572, Thermo Fisher Scientific) was applied at 1:200 dilution for one hour at room temperature. The nuclei were stained with DRAQ5^TM at 1:1000 dilution for 10 min at room temperature. After washing three times with PBS, the slides were mounted with fluorescence preserve VECTASHIELD^® HardSet^TM Antifade Mounting Medium (H-1400, Vector Laboratories, Eching, Germany). Images were taken with the Leica TCS SP8 confocal microscope.

Paraffin-embedded blocks were cut into 4-μm thick sections. The dewaxed and hydrated tissue sections were incubated with anti-PU.1 (Abcam, ab88082), anti-CD23 (Abcam, ab254162), anti-p-ERK (Abcam, ab229912), anti-CCL20 (Abcam, ab9829) and anti-IL-8 (Abcam, ab18672) antibodies for 2 h at room temperature and subsequently incubated with a goat-anti-rabbit antibody for 40 min. The degree of staining was determined with diaminobenzidine (DAB) chromogen (Bio-Rad, Inc., CA, USA) and detected with a microscope (Olympus, Japan).