Anti human caspase 3 antibody

Tissue sections 5 μm thick were deparaffinated, rehydrated and stained by immersing in GILL II Hematoxylin, followed by eosin staining. For the study of apoptotic nuclei, tissue sections were stained with DAPI Fluoromont-G (EMS, Madrid, Spain) for 10 min and detected in a fluorescence microscope (E600/E400, Nikon, Tokyo, Japan) equipped with a digital photography machine (DXM1200F, Nikon). The expression of activated caspase-3 was investigated by immunohistochemistry using a rabbit polyclonal anti-human caspase-3 antibody (Cell Signaling, Barcelona, Spain), which recognizes the active, cleaved caspase-3 form. For antigen retrieval, the sections were boiled in 10 mM citrate buffer, pH 6.0 for 30 min. After blocking with 5% horse serum diluted in PBS for 1 hr at room temperature, sections were incubated at 4 °C in humid chambers with the anti-caspase-3 antibody at 1/150 dilution for 1h followed by ready to use secondary anti-rabbit antibody (Vector Laboratories, Peterborough, UK) for 30 min. As a chromogenic substrate, DAB (Agilent, Madrid, Spain) was used, followed by hematoxylin counterstaining. Appropiate negative control stainings were also performed.

Precipitated materials, fibroblast lysates, conditioned media from fibroblast culture, mouse BALF, and mouse and human lung homogenates were analyzed by immunoblotting as previously described (9 (link)). The antibodies used were anti–human ENO-1 antibody (Abcam), anti–human uPAR antibody (MilliporeSigma), anti–human endostatin antibody to detect E4 peptide (R&D Systems), anti–human fibronectin antibody (Santa Cruz Biotechnology), anti–human collagen type I antibody (Abnova, Cedarlane, and CloudClone), anti–human GAPDH antibody (Santa Cruz Biotechnology), anti–human uPA antibody (Proteintech), anti–human PAI-1 antibody (Proteintech), anti–human MMP-1 antibody (Abcam), anti–human MMP-3 antibody (Abcam), anti–human caspase-3 antibody (Cell Signaling Technology), anti–human Egr-1 antibody (Cell Signaling Technology), anti–human β-actin antibody (Santa Cruz Biotechnology), anti–mouse uPA antibody (Abcam), anti–mouse PAI-1 antibody (Proteintech), anti–mouse HGF antibody (R&D Systems), anti–mouse collagen type 1 antibody (Southern Biotech), anti–mouse GAPDH antibody, and anti-V5 antibody (MilliporeSigma) as primary antibodies. Horseradish peroxidase–conjugated antibodies were used as a secondary antibody. Signals were detected using chemiluminescence.