Horseradish peroxidase conjugated rabbit anti goat

Brain samples were rapidly obtained from each mouse following decapitation and protein concentrations were determined with a BCA detection kit. Twenty micrograms of each brain lysate was loaded and separated by 4-20% SDS-polyacrylamide gradient gel electrophoresis and then transferred to 0.45 μm polyvinylidene fluoride membranes (100 V, 80 min). After blocking for 1 hour in PBST (10 mm sodium phosphate, 0.9% NaCl and 0.1% Tween 20) containing 5% non-fat dry milk, blots were incubated overnight at 4 °C with goat anti-mouse P2Y1, P2Y2, P2Y4, P2Y6, P2Y12, P2X7 and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies in PBST containing 5% non-fat milk. The blots were washed with PBST and incubated for 1 hour with horseradish peroxidase-conjugated rabbit anti-goat (Santa Cruz Biotechnology) diluted in PBST. Immunoreactivity of the protein bands were detected by enhanced chemiluminescent autoradiography (ECL kit, Amersham Pharmacia Biotech, Piscataway, NJ). A molecular weight standard (Bio-Rad Laboratories, Hercules, CA) was used to evaluate the molecular weight of detected Acta Biologica Hungarica 68, 2017 bands. The immunoblot bands were quantified through measurement of band intensity with ImageJ software using the same pixel scale for all pictures. Band intensities were normalized by β-actin expression and expressed as arbitrary units.