Maruyama H, Morino H, Ito H, Izumi Y, Kato H, Watanabe Y, Kinoshita Y, Kamada M, Nodera H, Suzuki H, Komure O, Matsuura S, Kobatake K, Morimoto N, Abe K, Suzuki N, Aoki M, Kawata A, Hirai T, Kato T, Ogasawara K, Hirano A, Takumi T, Kusaka H, Hagiwara K, Kaji R, Kawakami H. Mutations of optineurin in amyotrophic lateral sclerosis. Nature. 2010 May 13;465(7295):223-6. PubMed Abstract

REAGENTS/MATERIAL:
Immunofluorescence microscopy: The following antibodies were used: mouse monoclonal anti-GM130 (35) (BD Transduction Laboratories) and affinity-purified rabbit anti-Flag (Sigma, 1:1,000).
Western blotting: Polyclonal antibodies recognizing the carboxy (C)-terminal part of OPTN (Cayman Chemical) and anti-rabbit IgG-HRP (R&D Systems) were used. For the internal control, we used G3PDH polyclonal antibody (IMGENEX).
Immunohistochemistry: of mouse nervous tissue. Rabbit polyclonal antibodies raised against various peptides of human/mouse OPTN origin gave consistent and reasonable results. One such antibody was OPTN-C raised against the C-terminal part of OPTN, which is identical between human and mouse (amino acids 575–591) (Cayman Chemical).
Post-mortem material from one of the OPTN mutant cases (subject 5) was available. Sections (6 mm) of formalin-fixed, paraffin-embedded spinal cord were examined with Klu¨ver–Barrera and haematoxylin and eosin staining. Some sections stained with haematoxylin and eosin were photographed, decolourized and immunostained with OPTN-C or with rabbit OPTN-I (Cayman Chemical). In addition, lumbar spinal cord tissue was obtained from clinically and neuropathologically proven cases of SALS and familial ALS with the A4V SOD1 mutation. After confirmation of complete removal of the OPTN antibody, we immunostained the same sections with mouse monoclonal anti-ubiquitin (Santa Cruz Biotechnology) and rabbit anti-ubiquitin (Sigma Aldrich), with mouse monoclonal anti-TDP-43 (Abnova), rabbit polyclonal anti-TDP-43 (Proteintech Group), or with mouse monoclonal anti-SOD1 (Lab Vision Corporation) or rabbit polyclonal anti-SOD1 (Stressgen Biotechnologies).