After behavioral test, rats were deeply anesthetized with pentobarbital sodium (50 mg/kg). The spinal cord was removed quickly and the lumber segments of the dorsal horn (L4-L5) were punched (13 (link)) and homogenized in the lysis buffer. The total and nuclear protein were prepared and processed for immunoblotting. The following antibodies were used: polyclonal antibodies against NR2B (1:1000, Millipore), GluR1 (1:1000, Millipore), or monoclonal anti-β-actin antibody (1:1000; Santa Cruz Biotechnology), and the horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibody (1:10,000; Jackson ImmunoResearch Laboratories). The immunoreactivity was detected using enhanced chemiluminescence (ECL Advance Kit, Amersham Biosciences). The intensity of bands was analyzed quantitatively with ImageJ software.
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Quantitative Western Blot Analysis
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Actin
Anti igg
Antibodies
Antibody
Behavioral test
Buffer
Chemiluminescence
Dorsal horn
Horseradish peroxidase
Monoclonal antibody
Mouse
Nr2b
Nuclear protein
Pentobarbital sodium
Rabbit
Rats
Spinal cord
Corresponding Organization :
Other organizations : Qingdao University, Affiliated Hospital of Qingdao University
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Variable analysis
independent variables
- Pentobarbital sodium (50 mg/kg) used to deeply anesthetize the rats
- Total and nuclear protein levels of NR2B and GluR1 in the lumbar segments of the dorsal horn (L4-L5)
- Spinal cord removal
- Punching and homogenizing the lumbar segments of the dorsal horn (L4-L5)
- Preparation and processing of total and nuclear protein for immunoblotting
- Use of polyclonal antibodies against NR2B and GluR1, and monoclonal anti-β-actin antibody
- Use of horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibody
- Detection of immunoreactivity using enhanced chemiluminescence (ECL Advance Kit)
- Quantitative analysis of band intensity using ImageJ software
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