Ecl prime blocking agent

Cerebral cortex tissue were obtained from mice 3 hours after sham operation or CA/CPR and treated with hypothermia or maintained at normothermia. Tissue homogenates were centrifuged and the supernatant proteins were fractionated on Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA) and transferred to PVDF membranes. Membranes were blocked for 1 hour in 2% ECL Prime blocking agent (GE Healthcare, Piscataway, NJ) and incubated overnight with primary antibodies (Cell Signaling Technology Inc., Danvers, MA, unless otherwise noted) against NOS1 (1:5,000), NOS2 (1:5,000), total NOS3 (1:5,000; BD Biosciences, San Jose, CA), phospho NOS3 at Ser¹¹⁷⁷ (1:1,000), phosho NOS3 at Thr⁴⁹⁵ (1:5,000; BD Biosciences), total (1;1,000) and phospho Akt at Ser⁴⁷³ (1;1,000) and Ser³⁰⁸ (1;1,000), total (1;1,000) and phoshpho 5'-prime-Amp-activated protein kinase α (AMPKα) at Thr¹⁷² (1;1,000), total (1;1,000) and phospho VASP at Ser²³⁹ (1;1,000), Heat shock protein 90 (Hsp90, 1;10,000), and β-tubulin (1:5,000). Bound antibody was detected with a horseradish peroxidase-linked antibody directed against rabbit IgG (1:5,000) or mouse IgG (1:20,000; Thermo-Pierce, Rockford, IL) and was visualized using chemiluminescence with Lumigen TMA-6 (Lumigen, Inc., Southfield, MI) or Immobilon Western (Millipore, Billerica, MA).

Dot blot assays were performed as previously described (26 (link)). Briefly, 1 ml aliquots of LL-Wt and LL-Gag cultures cultured overnight were washed twice with PBS and resuspended in 100 μl of PBS; 5 μl of these preparations were spotted onto a nitrocellulose membrane and air-dried. After blocking for 1 hour at room temperature with 2% ECL prime blocking agent (GE Healthcare) in PBS + 0.05% Tween 20 (PBST), the membranes were incubated with a 1:4,000 dilution of a rabbit anti-T3 hyper immune serum or a 1:5,000 dilution of a mouse anti-Gag monoclonal antibody (clone H12.5c, NIH AIDS reagent program;). Membranes were then washed with PBST and incubated with a 1:10,000 dilution of anti-rabbit IgG-HRP (Southern Biotech, Birmingham, Alabama, USA) (anti-T3 antibody treated blots) or anti-mouse IgG-HRP (Southern Biotech, Birmingham, Alabama, USA) (anti-Gag antibody treated blots) at 1:10,000 dilutions. The presence of bound antibodies was detected using AEC substrate (BD Biosciences, San Jones, CA, USA).