Propofol

The salts used in buffers, alfaxalone, diazepam, CNQX and DL-APV used in electrophysiological experiments were bought from Sigma-Aldrich (St. Louis, MO). Propofol was purchased from MP Biomedicals (Solon, OH). Stock solution of alfaxalone was made in DMSO at 10 mM and stored at room temperature. Stock solution of Propofol was made in DMSO at 200 mM and stored at room temperature. For electrophysiological recordings, stock solution of diazepam was made in ND96 at 30 μM and stored at −20 °C. Dilutions to lower concentrations were made from that solution. The highest final concentration of DMSO was 0.3% (v/v). This concentration of DMSO has been shown to be without effect on currents from recombinant α1β2γ2L GABA_A receptors or GABA_A receptor-mediated synaptic currents^{52 (link),53 (link)}.
Pharmaceutical grade reagents were used in all behavioral experiments. Propofol (10 mg/ml, Hospira, Inc., San Jose, CA), alfaxalone (10 mg/ml, Jurox, Inc., Kansas City, MO), diazepam (5 mg/ml, Hospira, Inc.), combinations of either Propofol and alfaxalone, or diazepam and alfaxalone, or sterile normal saline (20 Units) were injected intraperitoneally using U-100 insulin syringes. diazepam was diluted to 0.5 mg/ml with normal saline prior to injection.

The salts and HEPES used to prepare the bath solution, GABA, and 3α5αP were purchased from Sigma-Aldrich (St. Louis, MO). Propofol was purchased from MP Biomedicals (Solon, OH). Pregnenolone sulfate (PS) was bought from Tocris (Bio-Techne, Minneapolis, MN).
The stock solution of GABA was made in the bath solution at 500 mM, stored in aliquots at −20 °C, and diluted on the day of experiment. Stock solution of Propofol was made in DMSO at 200 mM and stored at room temperature. 3α5αP was dissolved in DMSO at 10–20 mM and stored at room temperature. PS was dissolved in DMSO at 50 mM and stored at 4 °C.

The inorganic salts and HEPES used to prepare the bath solution were purchased from Sigma‐Aldrich (St. Louis, MO). GABA was purchased from Sigma‐Aldrich, solubilized in bath solution at 500 mmol/L, and stored in aliquots at −20°C. Taurine was purchased from Sigma‐Aldrich, solubilized in bath solution at 300 mmol/L, pH‐adjusted, and diluted as needed. Propofol was purchased from MP Biomedicals (Solon, OH). The stock solution of Propofol was made in DMSO at 200 mmol/L and stored at room temperature. The steroid 3α5αP was bought from Sigma‐Aldrich or Tocris (Bio‐Techne, Minneapolis, MN), dissolved in DMSO at 10–20 mmol/L and stored at room temperature.

Primary antibodies used in this study are listed in Table 1. Peroxidase-conjugated anti-mouse and anti-rabbit IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The enhanced chemiluminescence (ECL) kit was from Pierce (Rockford, IL, USA). The ABC staining system was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Propofol was purchased from MP Biomedicals (Solon, OH, USA). Insulin (Humulin R U-100) was from Eli Lily (Indianapolis, IN, USA). Other chemicals were from Sigma-Aldrich (St. Louis, MO, USA).

Primary antibodies used in this study are listed in Table 1. Peroxidase-conjugated anti-mouse and anti-rabbit IgG were obtained from Jackson Immuno Research Laboratories (West Grove, PA, USA). The enhanced chemiluminescence (ECL) kit was from Pierce (Rockford, IL, USA). The ABC staining system was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Propofol was purchased from MP Biomedicals (Solon, OH, USA). Insulin (Humulin R U-100) was from Eli Lily (Indianapolis, IN, USA). Other chemicals were from Sigma-Aldrich (St. Louis, MO, USA).

Sevoflurane and isoflurane were purchased from Henry Schein, Inc. (Melville, NY, USA). Propofol was purchased from MP Biomedicals (Solon, OH, USA). Other chemicals were from Sigma-Aldrich (St. Louis, MO, USA).

The DXM standard (purity 99.9%) was purchased from Santa Cruz Biotechnology, and cyclophosphamide monohydrate (internal standard) from MP Biomedicals. (Fountain Pkwy, Solon OH, USA). Acetonitrile and methanol HPLC grade were obtained from EMD Millipore Co (Mexico). Ethyl acetate and formic acid reagent grade were purchased from Merck (Darmstat, Germany). Dichloroethane from Electron Microscopy Sciences (Washington, DC, USA).
The standards for selectivity were: ropivacaine, propofol, acetaminophen, fentanyl, which were purchased from MP Biomedicals (Fountain Pkwy, Solon OH, USA) and Toronto Research Chemicals Inc (Toronto, Canada). Milli-Q grade water (Millipore, Molsheim, France) was used to prepare all solutions and dilutions. Erythrocyte fraction (kept at 4°C) and blank plasma (frozen at -80°C), were obtained from the blood reservoir of our institution (healthy adult donors), having citrate phosphate dextrose as anticoagulant.