Rc dc reagent kit

Immediately after sacrifice, brains were removed and the hippocampus was isolated. Hippocampal slices (600 μm) were prepared using a McIlwan tissue chopper to facilitate microdissection of hippocampal regions as follows: first, the CA3 region was separated from CA1 and DG to then separate the CA1 from the DG (Silva et al., 2001 (link)). The pieces of tissue for each region taken from the same rat were pooled in one sample, quickly frozen on dry ice and stored at-80°C until use. Tissue lysates were prepared by briefly sonicating the pieces of tissue in RIPA buffer containing protease and phosphatase inhibitors. Lysates were cleared of cellular debris by centrifugation at 17,000 X g for 20 min. Protein concentration was determined using the Bio-Rad RC/DC reagent kit (Bio-Rad Laboratories, Hercules, CA, USA).

Neurons were cultured from rat hippocampal tissue at postnatal day 0–2 (P0–P2), as described (Gonzalez, 2014 (link)). Briefly, hippocampal tissue was dissociated with papain by mechanical trituration and plated in MEM media supplemented with 10% fetal bovine serum (4 ml at 400,000 cells/ml). After one day in culture, cells were switched to Neurobasal-A media supplemented with B27 and inhibitors of glial proliferation. Cultures were maintained at 37°C and 5% CO₂ and used after 14–15 days in vitro (DIV). To study calpain-dependent proteolysis, cells were incubated with glutamate plus glycine (100 μM each in HBSS) for 30 min and returned to conditioned culture media for up to six hours. To analyze calpain inhibition, cells were stimulated the presence of DMSO or MDL-28170 (100 μM). Cell lysates were prepared in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined using the Bio-Rad RC/DC reagent kit (Bio-Rad Laboratories, Hercules, CA, USA).

Following the rapid isolation of hippocampus from the rest of the brain, hippocampal slices (600 μm) were prepared using a McIlwan tissue chopper. Each individual slice was then microdissected to isolate the Cornus Ammonis 1 (CA1); the region of hippocampus where more prominent levels of calpain activation and neuronal death can be detected after SE (Araujo et al., 2008 (link)). For microdissection, the CA3 region was separated from the CA1 and DG; then, the CA1 region was separated from DG through the hippocampal sulcus (Silva et al., 2001 (link)). All CA1 pieces collected from the same rat were pooled together, frozen on dry ice and stored at −80°C. Whole tissue lysates were prepared by brief tissue sonication in RIPA buffer containing a mixture of protease and phosphatase inhibitors. To remove cell debris, lysates were cleared by centrifugation at 17,000 X g for 20 min. Protein concentration was determined using the Bio-Rad RC/DC reagent kit (Bio-Rad Laboratories, Hercules, CA, USA).