Abc staining system kit

The NMDA receptor is primarily composed of two NR1 subunits and two NR2A or NR2B subunits.18 (link) The NR1 subunit determines the function of the NMDA receptor.19 (link) Rats were anesthetized by intraperitoneal injection of sodium 100 mg kg⁻¹ pentobarbitone and then perfused transcardially with 0.01 mol l⁻¹ phosphate-buffered saline (PBS) followed by 4% paraformaldehyde. Thirty-micron coronal brain sections were cut with a freezing microtome (CM 1900-1-1, Leica, Frankfurt, Germany) and then immersed in cryoprotectant solution (30% sucrose, 30% ethylene glycol in 0.1 mol l⁻¹ phosphate buffer, and 0.01% sodium azide) and stored at −20°C until further processing. Sections were incubated with a rabbit anti-NR1 antibody (Sigma, USA, G0541) diluted in 0.01 mol l⁻¹ PBS at 4°C overnight. Sections were further incubated with a biotinylated secondary antibody in incubation solution from the ABC staining system kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min at 37°C according to the manufacturer's instructions. NR1-positive neurons in the PVN were observed with a conventional light microscope (DP70, Olympus, Tokyo, Japan). PVN sites were identified according to the rat brain atlas of Paxinos and Watson.20 We observed neurons with NR1-like immunoreactivity in four PVN sections and averaged the numbers as the final result for each animal.