Pentothal sodium

For BrdU immunohistochemistry, animals were deeply anesthetized by i.v.injection of Thiopentone sodium (100 mg/kg body weight) (Pentothal sodium, Abbott laboratories) and transcardially perfused with normal saline and then with 4% paraformaldehyde in phosphate buffered saline (PBS, 0.1 M, pH 7.4). After post fixation of 24 h of the brain in paraformaldehyde/PBS solution, microtome sections of 5 µm thick was cut coronally kept onto Poly-L-lysine coated glass slides and processed for immunohistochemistry (IHC). For IHC, tissue sections were rehydrated and treated with 0.1 M citrate buffer, pH 6.0 for 10 min in microwave oven and rinsed with PBS containing 0.05% Tween-20 (TBST). Sections were then incubated with 1 M HCl at 54 °C for 45 min followed by TBST washing. Sections were incubated with 0.3% H₂O₂ for 10 min and washed by TBST. Sections were incubated with 5% BSA in TBST, followed by overnight incubation with mouse monoclonal anti-BrdU antibody (CST, Bu20a) diluted in blocking buffer (CST, 1:200) at 4 °C. After being washed with TBST, sections were incubated with Alexa flour-conjugated goat anti-mouse (Abcam, ab150113) secondary antibody diluted in blocking buffer for 1 h at RT and section were counterstained with DAPI for 30 min.

Drugs used in this study were purchased from the source specified: pentothal sodium (Abbott Laboratories, Pakistan), isoproterenol hydrochloride, atropine sulfate, phenylephrine hydrochloride, potassium chloride, atenolol, and Nω-nitro-L-arginine methyl ester hydrochloride (_L-NAME) (Sigma Chemicals Company, USA). Analytical grade chemicals were used. All chemical stock solutions were made in distilled water and normal saline.