Mouse anti hif 1α antibody

Paraffin (Paraplast Tissue Embedding Medium REF 501006) was purchased from McCormick Scientific (St. Louis, MO) and hematoxylin from Merck (Germany). Hypoxyprobe-^TM1-Kit was obtained from HPI (Burlington, USA). Protein block and polyclonal rabbit anti-mouse immunoglobulins were from DAKO (Denmark). H₂O₂ was from Roth (Germany), Tween 20 from Sigma-Aldrich (Germany), ABC kit from Vector Laboratories (Burlington, USA), 3′,3′-diaminobenzidine (DAB) from Thermo Fisher Scientific (Fremont, USA), and mouse anti-HIF-1α antibody from Novus Biologicals (Cambridge, UK). Isoflurane (Florene) was obtained from Abbott (Wiesbaden, Germany), ketamine 10% from Ceva (Düsseldorf, Germany), lidocaine (Xylocain 1%) from AstraZeneca (Wedel, Germany), and Ringer's solution Macoflex N from MacoPharma International (Langen, Germany). Portex catheters (0.58 mm i.d., 0.96 mm o.d.) were purchased from Smiths Medical International (Hythe, UK) and medical oxygen was from Air Liquide (Düsseldorf, Germany).

Immunohistochemistry was performed on paraformaldehyde-fixed-paraffin-embedded samples according to the manufacturer's instructions (Vector Laboratories, USA; compare also Scheerer et al. [22] (link)). As primary antibody a mouse anti-HIF-1α antibody (dilution 1∶10,000, Novus Biologicals) was used. For detection of HIF-1α a catalyzed signal amplification system (DAKO, Denmark) were used. Specific staining was visualized by incubation with 3′,3′-diaminobenzidine (Vector Laboratories). Hematoxylin was used for counter-staining.

Lung tissue was homogenized and total protein collected using the PARIS kit (Ambion, Austin, TX) as previously described (Farrow et al., 2008a (link)). Protein lysates from PASMC were prepared using 1X Mg-lysis buffer (Upstate, Charlottesville, VA) supplemented with a protease inhibitor cocktail (Sigma). Protein concentration was measured using the Bradford method (Bradford, 1976 (link)). Total protein (40 μg) was separated on a 4–20% SDS-polyacrylamide gel (Biorad, Hercules, CA) and then transferred to a nitrocellulose membrane (Amersham, Arlington Heights, IL). Western blot was then performed as previously described (Farrow et al., 2008a (link),b (link)). Briefly, membranes were blocked at room temperature with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 (1X TBST) and were then incubated overnight at 4°C with a mouse anti-HIF-1α antibody (Novus Biologicals, Littleton, CO) in 5% milk + 1X TBST at a 1:1000 dilution. The membranes were washed and incubated with an anti-mouse secondary antibody conjugated to horseradish peroxidase (Pierce, Rockford, IL) diluted 1:1000 in 5% milk + 1X TBST. Membranes were washed and exposed via chemiluminescence (Pierce). Bands were analyzed using a Digital Science Image Station (Kodak, Rochester, NY). Expression within each Western blot was normalized to β-actin.