Rabbit anti rip3 antibody

Rabbit anti-RIP3 antibody, Nec-1, and propidium iodide (PI) were obtained from Sigma-Aldrich (St Louis, MO, USA), rabbit anti-β-tubulin was from Abcam (Cambridge, UK), and the fluorescein isothiocyanate-Annexin V/PI apoptosis assay kit was from Clontech (Mountain View, CA, USA). Morpholino oligonucleotides were synthesized by Gene Tools, LLC (Philomath, OR, USA). Bicinchoninic acid assay was purchased from Pierce (Rockford, IL, USA). Lipid peroxidation (MDA) was obtained from Jian-Cheng Biotechnical Co. (Nanjing, Jiangsu, China). Goat anti-rabbit secondary antibody was obtained from Jackson Immuno Research Inc. (Lancaster, PA, USA).

In order to examine the expression of RIP3 in a specific population of cells, the transverse sections obtained 3 days after hemisection were double-stained for RIP3 and various cell type markers: NeuN for neurons, GFAP for astrocytes and Olig2 for oligodendrocytes. The sections were incubated with a mixture of rabbit anti-RIP3 antibody (1:100; Sigma-Aldrich) and either goat anti-Olig2 (1:100; Santa Cruz Biotechnology), mouse anti-GFAP (1:50; Dako) or mouse anti-NeuN (1:100; Chemicon) antibodies diluted in PBS overnight at 4 °C. After rinsing with PBS, the sections were incubated with secondary antibodies, and then mounted with Vectashield containing DAPI to label the nuclei (Vector Laboratories). The specificity of the RIP3 antibody was evaluated by omission of the primary antibody in immunohistochemistry. Omitting the primary antibody (no primary immunoglobulin G control) in this protocol abrogated the staining, demonstrating the specificity of the immunoreactive staining [14 (link), 24 (link)].