Western blot analysis was conducted following previous reports.36 (link)–38 (link) The rat brain tissue was homogenized in protein lysate buffer. The homogenate was subjected to 10% SDS-PAGE and then electrophoretically transferred to a nitrocellulose membrane. After blocking with 5% skim milk in TBST for 1 h, the membrane was incubated with primary antibody (against active GFAP, ICAM-1, and caspase-3) at 4°C overnight and subsequently treated with alkaline phosphatase-conjugated secondary antibodies. GFAP, ICAM-1, and caspase-3 were developed using BCIP/NBT. Blots were stained with anti-β-actin antibody, and the quantification of proteins was normalized based on β-actin band density. The antigen–antibody products were detected with Thermo Scientific Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA). The results were analyzed with a FluorChem™ system (Alpha Innotech, San Leandro, CA, USA).
Protocol detail
Quantifying GFAP, ICAM-1, and Caspase-3 in Rat Brain
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Actin
Alkaline phosphatase
Anti antibody
Antibodies
Antibody
Antigen
Brain
Buffer
Caspase 3
Gfap
Icam 1
Membrane
Milk
Nitrocellulose
Proteins
Sds page
Tissue
Western blot analysis
Corresponding Organization :
Other organizations : Shantou University, Shantou University Medical College, Jiaxing University, University of Maryland, College Park, Zhejiang Chinese Medical University
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Variable analysis
independent variables
- None explicitly mentioned
- Levels of active GFAP
- Levels of ICAM-1
- Levels of caspase-3
- Rat brain tissue
- Protein lysate buffer
- 10% SDS-PAGE
- Nitrocellulose membrane
- 5% skim milk in TBST for 1 h blocking
- Primary antibody (against active GFAP, ICAM-1, and caspase-3) at 4°C overnight
- Alkaline phosphatase-conjugated secondary antibodies
- BCIP/NBT for development
- Anti-β-actin antibody for normalization
- Thermo Scientific Super Signal West Pico Chemiluminescent Substrate
- FluorChem™ system for analysis
- None explicitly mentioned
- None explicitly mentioned
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