Journal: PLoS ONE
Article Title: Substance P Inhibits Hyperosmotic Stress-Induced Apoptosis in Corneal Epithelial Cells through the Mechanism of Akt Activation and Reactive Oxygen Species Scavenging via the Neurokinin-1 Receptor
Figure Lengend Snippet: Role of NK-1 receptor in the anti-apoptotic effects of SP. Mouse corneal epithelial cells were treated with 1 μM NK-1 receptor antagonist L-733,060 with 1 μM SP 2 h before the addition of glucose for 24 h. The phosphorylation of Akt was evaluated by Immunofluorescence staining or Western blot (A). The apoptosis was evaluated by FACS analysis followed by FITC-Annexin V/PI staining (B), and the detection of Bad, Bax, AIF, Ca 2+ and mitochondrial membrane potential (C). The intracellular ROS and glutathione (GSH) were detected by staining with the fluorescence probes (D) and measured by the fluorescence intensity (E). The cellular total antioxidant capacity (TAC) was measured by the ABTS assay (E).
Article Snippet: Immunofluorescence staining Cells were fixed by 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 5 min and blocked with 5% bovine serum albumin (BSA) for 1 h. The samples were stained with primary antibodies against AIF (Santa Cruz Biotechnology, Santa Cruz, CA), p-Akt (pS473, Epitomics, Burlingame, CA), MnSOD, catalase, NQO1, Hmox1, Bad and Bax (abcam, Cambridge, MA) at a concentration of 1:100 overnight at 4°C, and then incubated with fluorescein-conjugated secondary antibodies (Santa Cruz Biotechnology) at 37°C for 30 min.
Techniques: Immunofluorescence, Staining, Western Blot, FACS, Fluorescence, ABTS Assay