Anti albumin

At indicated differentiation times, culture cells were washed with PBS twice and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. Then, cells were permeabilized with 0.1% Triton-X twice for 10 min, blocked with 2% BSA and incubated overnight with primary antibodies, including anti-SSEA-4 (mouse, 1:100, Cell Signaling), anti-albumin (mouse, 1:100, Santa Cruz Biotechnology), anti-Ki-67 (rabbit, 1:100, Millipore), anti-p21 (rabbit, 1:100, Santa Cruz Biotechnology) and anti-P-ERK (rabbit, 1:100, Cell Signaling). No permeabilization was performed for SSEA-4 detection. Secondary antibodies including FITC-conjugated anti-mouse IgG (goat, 1:100, Sigma) and Alexa Fluor 488-conjugated anti-rabbit IgG (goat, 1:100, Thermo Fisher Scientific) were used according to the manufacturer’s instructions. The samples were incubated at room temperature for 1 h. After further washings with PBS and counterstained with DAPI, samples were mounting with prolong Gold antifading solution (Thermoscientific). In negative controls, primary antibody was omitted for each respective fluorescent tracer (data not shown). Cells were visualized and photomicrographed under an inverted fluorescence microscope (Nikon). Positive FITC or Alexa 488 cells were analyzed and quantified using FIJI-Image J software (Bethesda, MD, USA)

Immunoblotting and co-immunoprecipitation studies were carried out as previously described^{19 (link)}. Briefly, blood serum samples (30 μg) were prepared from animals in seven groups mentioned in a previous section (Sham, LPS, MCAO, MCAO+LPS, and MCAO+LPS/A box, MCAO+LPS/HPep1, MCAO+LPS+LPS-RS) and separated in 10% sodium dodecyl sulfate-polyacrylamide gels. After blocking membranes with 5% non-fat milk for 1 h, they were incubated with primary antibodies for anti-HMGB1 (1:2000; Abcam, Cambridge, UK; ab67281), anti-albumin (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA; SC-374670), and anti-α-tubulin (1:5000; Cell Signaling, Danvers, MA, USA; #2144) overnight at 4 °C. The next day, blots were detected using anti-rabbit or anti-mouse horse radish peroxidase-conjugated secondary antibody (1:2000; Santa Cruz Biotechnology; SC-2004 or SC-2005) and a Chemiluminescence Kit (Roche, Basel, Switzerland). Blood sera containing 500 μg of protein were immunoprecipitated with 2 μl of anti-LPS antibody (Abcam, Cambridge, UK; ab35654) overnight at 4℃. Pre-equilibrated protein A PLUS-Agarose beads (Pierce Biotechnology, Rockford, IL, USA; #20365) were then added and incubated for 2 h at 4 °C on a rotating wheel. Beads were washed three times with radioimmunoprecipitation assay buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.