Rabbit polyclonal antibody against mpo

Immunostaining for AnxA1, myeloperoxidase (MPO), CD45, Ly-6C, Ly-6G, Gal-3, FPR1, FPR2, and cleaved caspase-3 was performed on 5-μm-thick sections of rat lung tissue. Briefly, formalin-fixed paraffin lung tissue sections were deparaffinized before antigen retrieval. The endogenous peroxidases in lung tissue were quenched with 3% H₂O₂ and 100% methanol for 15 min. The sections were immunostained with a rabbit polyclonal antibody against AnxA1 (1:100, Bioss, USA), a rabbit polyclonal antibody against MPO (1:200, Thermo Fisher Scientific, USA), a mouse monoclonal antibody against CD45 (1:100, Merck KGaA, Germany), a mouse monoclonal antibody against Ly6C (1:200, Santa Cruz Biotechnology), a rabbit polyclonal antibody against Ly6G (1:300, Biorbyt, UK), a mouse monoclonal antibody against Gal-3 (1:100, Santa Cruz Biotechnology), and a rabbit polyclonal anti-cleaved caspase-3 antibody (1:200, CST, USA). Sections were washed twice with PBS containing 0.1% Tween-20 (PBST) and then incubated with a rat-specific horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min. The HRP signal was visualized after a chromogenic reaction with diaminobenzidine for 5 min, and the lung tissue sections were counterstained with hematoxylin. Subsequently, the slides were dehydrated in a gradient of alcohol solutions (16 (link)).

Histologic and immunohistochemical examinations were carried out as previously described^{35 (link)–37 (link)}. In brief, 6 or 24 h after cold-warm-cycles, rat right middle lung lobe was excised, fixed in 4% paraformaldehyde in 0.1 M phosphate buffer solution (pH 7.4), and processed for paraffin sections using an automated processing unit (RM2255, Leica, Berlin, Germany). The sections of 5 μm of pulmonary specimens for histology were stained by hematoxylin and eosin (H&E). The images were captured by a digital camera connected to a microscope (BX512DP70, Olympus, Tokyo, Japan). The sections for immunohistochemistry were incubated with rabbit polyclonal antibody against MPO (1:200, Thermo Scientific, Fremont, CA, USA), rabbit antibody against CD68 (1:50, Abcam, Cambridge, UK) or mouse antibody against Claudin-5 (1:50, Santa Cruz Biotechnology, Santa Cruz, USA) after blocked with bovine serum albumin, and then incubated with a biotinylated secondary antibody from an avidin-biotin-complex-peroxidase kit or a fluorescent secondary antibody. Positive staining was revealed by reacting with diaminobenzidine (BD Biosciences Pharmingen, CA, USA) or a laser scanning confocal microscope (TCS SP5, Leica, Mannheim, Germany).