Collection of tissue samples from the hippocampus At predetermined time intervals after microinjection of KA or PBS into the hippocampus, rats were Crizotinib 877399-52-5 again anesthetized by ip admin istration of chloral hydrate and were per fused intracardially with 50 mL of warm saline Inhibitors,Modulators,Libraries that contained heparin. As we reported pre viously, the brain was rapidly removed under visual inspection and placed on a piece of gauze mois tened with ice cold 0. 9% saline. We routinely collected tissues from the ipsilateral and the contralateral hippocampal CA3 subfield. Hippocampal samples were stored at ?80 C until biochemical analysis. In experi ments involving immunofluorescence staining, brains were post fixed in 4% paraformaldehyde for 48 h at 4 C followed by cryoprotection with 30% sucrose solution.
The concentration of total proteins extracted from tissue samples was determined by the bicinchoninic acid protein assay. In some Inhibitors,Modulators,Libraries experiments, proteins from the nuclear or cyto solic fraction of the hippocampal samples were extracted by a commercial kit. Measurement of protein oxidation Oxidized protein was determined using a protein oxida tion detection kit. This kit provides reagents for sensitive immuno detection of the carbonyl group, which is a hallmark of the oxidation status of proteins. Total proteins extracted from the hippocampal CA3 subfield were reacted with 2,4 dinitrophenylhydrazine and derivatized to 2,4 dinitrophenylhydrazone. The DNP derivatized protein Inhibitors,Modulators,Libraries samples were separated on a 15% SDS polyacrylamide gel followed by western blot.
The blot was incubated with a rabbit Inhibitors,Modulators,Libraries anti DNP antibody, followed by incubation with a horseradish peroxidase conjugated goat anti rabbit IgG according to manufac turers instructions. Western blot analysis Western blot analysis for UCP2, PPAR��, Bcl 2, Bax, cytochrome c or B actin was carried out on proteins extracted from nuclear fractions or from mitochondrial or cytosolic fractions of hippocampal samples. The pur ity of the mitochondrial fraction was verified by the se lective expression of the mitochondrial inner membrane specific protein, cytochrome c oxidase subunit IV. Protein concentration was determined by the BCA Protein Assay. The primary antisera used included rabbit polyclonal antiserum against Bax and COX IV, goat polyclonal antiserum against UCP2, mouse monoclonal antiserum against Bcl 2, cytochrome c and PPAR�� or B actin.
B actin was used for internal control of total protein or proteins in the cytosolic fraction, and COX IV for proteins in the mito chondrial fraction. The secondary antisera used included horseradish peroxidase conjugated sheep anti mouse IgG for Bcl 2, cytochrome c, PPAR�� Inhibitors,Modulators,Libraries and B actin, donkey anti goat IgG for UCP2, or donkey 17-AAG price anti rabbit IgG for Bax, and COX IV. Specific antibody antigen complex was detected by an enhanced chemiluminescence western blot detection system.