Rabbit anti human ho 1 antibody

For immunohistochemistry, rabbit anti-human cleaved caspase 3 antibody (1:300, Cell Signaling Technology, Danvers, MA), rabbit anti-human hypoxia-inducible factor 1a antibody (1:1000, Novus Biologicals, Littleton, CO), anti-human 4-hydroxy-2-nonenal antibody (10 µg/ml, Japan Institute for the Control of Aging, Japan), rabbit anti-human HO-1 antibody (1:500, Enzo Life Sciences, Farmingdale, NY), rabbit anti-human fibrin/fibrinogen antibody (1:4000, Dako, Denmark), and rat anti-mouse MOMA2 antibody (1:400, AbD Serotec, Raleigh, NC) were used. TUNEL stain kit was from Wako chemicals (Osaka, Japan). Neutrophils were visualized using Naphthol AS-D chloroacetate Esterase stain (Muto Pure Chemicals, Tokyo, Japan). About 5 consecutive fields were examined in each slide at 100 or 200-fold magnification. All assessments were performed with ImageJ (National Institutes of Health, Bethesda, MD).

For western blot analysis, 20 μg of proteins was transferred into a 12% polyacrylamide gel and a SDS-PAGE in denaturizing conditions was performed at 100 V. After the electrophoresis, proteins were transferred into PVDF membranes in transfer buffer containing 20% methanol (v/v), 0.19 M glycine, and 0.025 M Tris-base of pH 8.3. For protein detection, membranes were incubated overnight (ON) with a specific rabbit anti-human HO-1 antibody (Enzo Life Science, Lörrach, Germany). After three washing steps with TBST (TBS with 0.5% Tween) for 5 min each, the membranes were incubated with an anti-rabbit HRP-conjugated (Thermo Fisher Scientific, Schwerte, Germany) antibody diluted 1:2000 for 1 h at RT and then with avidin–horseradish peroxidase complex (ABC complex, Biozol, Eching, Germany). GAPDH was used as loading control in the same gel and applied together with the HO-1 antibody. The chemiluminescence signal was generated by using luminol (A8511-5G, Sigma-Aldrich), 4-hydroxycinnamic acid (p-coumaric acid; C9008-25G, Sigma-Aldrich), and hydrogen peroxide (Merck, Darmstadt, Germany). The intensity of the bands was quantified by using the GeneSnap^® Software, Version 4.01c from Syngene, Darmstadt, Germany.