Nih imagej software

Equal volumes of serum from randomly chosen samples were heated with sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris–HCl, pH 6.8, 8% 2-mercaptoethanol). They were then electrophoresed on Criterion Stain-free 4–20% SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes at 100 V for 30 min. The membranes were blocked with PBS containing 5% nonfat dry milk and probed separately with each of the antibodies: anti-CKM (mouse monoclonal, 1:1,000, Santa Cruz, sc-365046), anti-COMP (mouse monoclonal, 1:1,000, Santa Cruz, sc374660), anti-EFEMP1 (mouse monoclonal, 1:1,000, Santa Cruz, sc-33722, 1:1,000), anti-FBLN1 (mouse monoclonal, 1:1,000, Santa Cruz, sc25281), anti-GSN (mouse monoclonal, 1:1,000, Abcam, ab11081), anti-PROS1 (rabbit monoclonal, 1:1,000, Santa Cruz, sc-52720). The membranes were washed three times in PBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Signals were detected using enhanced chemiluminescence and Gel Doc System (Bio-Rad). The blots were stripped and probed for the housekeeper protein transferrin. The signal intensity was quantified using Image Lab (Bio-Rad) and NIH ImageJ software (v1.45s).

Equal volumes of fasted plasmas were separated on SDS–PAGE gels and transferred onto 0.2 μM nitrocellulose membranes at 75 volts for 30 min. Membranes were blocked with Tris buffered saline (TBS) containing 5% non-fat dry milk and probed with anti-apoAI antibody (rabbit polyclonal 1:5,000 Merck #178422) and anti-apoC-I antibody (rabbit monoclonal 1:1,000 Abcam #ab198288) overnight at 4°C. They were then washed three times in TBS containing 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Signals were detected using enhanced chemiluminescence and Gel Doc System (Bio-Rad). The signal intensity was quantified using Image Lab (Bio-Rad) and NIH ImageJ software (v1.45s). Plasma PON1 (Cloud-Clone, #SEA243Hu) and SERPINA1 (Abcam, #ab108799) were measured by ELISA following the manufacturer’s protocols.

Hippocampal tissues and bEnd.3 cells were homogenized in RIPA lysis buffer containing 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1.5 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The samples were centrifuged at 12,000×g (4 °C) for 20 min, and the supernatants were harvested. Protein concentration was determined using a BCA kit. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF microporous membrane (Millipore, USA). The membranes were incubated with 5% non-fat milk at room temperature for 1 h and incubated overnight at 4 °C with primary antibodies: polyclonal rabbit anti-IL-17A receptor (1:500), monoclonal mouse anti-MMP-2 (1:1000), monoclonal mouse anti-MMP-9 (1:500), monoclonal mouse anti-claudin-5 (1:500) and monoclonal rabbit anti-occludin (1:1000). An antibody against GAPDH (1:1000) was also used as an internal standard. The membranes were incubated with goat anti-mouse secondary or goat anti-rabbit secondary antibodies at room temperature for 1 h and then detected with a chemiluminescent substrate. The relative densities of the protein bands were visualized and analysed by Image Lab software (Bio-Rad, Richmond, CA, USA) and NIH ImageJ software (Bethesda, MD, USA), respectively.