Electrophoresis gel imaging system

Six rats from each group were euthanized 48 hours post-occlusion. The brains were carefully removed and placed in chilled saline. Total protein was isolated directly from the ischemic penumbra after cell lysis and then added to Laemmli buffer (75 mM Tris-HCl containing 2% sodium dodecyl sulfate (SDS), 10% glycerol, 2% 2-mercaptoethanol, 0.002% bromphenol blue). The samples were heated to 95°C for 10 minutes and then separated on 10% Tris/glycine/SDS acrylamide gels (Bio-Rad, Hercules, CA, USA). The proteins were subsequently transferred to polyvinylidene difluoride (Minipore, Billerica, MA, USA) membranes and blocked for 2 hours at room temperature in 5% non-fat milk. The immunoblots were incubated for 2 hours at 37°C with mouse anti-occludin or mouse anti-ZO-1, both with β-actin monoclonal antibody (serving as the control) (all at 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing (3 × with Tris-buffered saline/0.05% Tween-20), the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (1:1,000; Santa Cruz Biotechnology) for 1 hour at 37°C. The membranes were then stained with tetrazotized o-dianisidine and β-naphthyl acid phosphate and analyzed with an electrophoresis gel imaging system (Bio-Rad) to obtain OD values. The levels of occludin and ZO-1 were then expressed as percentages relative to β-actin.

The levels of Notch receptors (Notch 1–3) and Delta-like (DLL)-4 in lung tissues were detected by RT-PCR. Total RNA of lung tissues was extracted using Trizol reagent (Takara, Dalian). We used 1 μL of cDNA with SYBR Green Master to configure the reaction system for PCR reaction. PCR amplifications were performed at 95°C for 5 min, followed by 30 cycles of thermal cycling at 95°C for 20 s, 60°C for 30 s, and 72°C for 30 s. The results were observed and photographed in an electrophoresis gel imaging system (Bio-Rad, USA). Each band value relative to the reference gene (β-actin) was calculated and analyzed using Image J software, and then normalized to compare the expression levels of Notch1, 2, and 3 and DLL4 mRNA in tissues. The primers used in this study were: