Rabbit anti ps6

Rats were anesthetized using 250 mg/kg ketamine and 2% xylazine (10 mg/kg, Sigma-Aldrich) for a duration of 6, 12 or 24 h following surgery, and were subsequently sacrificed by rapid decapitation. The collected brain tissues were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 8 h at 0°C. Fixed brains were paraffin embedded and cut into 2 µm sections using a microtome (Leica Microsystems GmbH, Wetzlar, Germany). Sections were briefly washed with TBS and incubated for 30 min in 3% H2O2 to quench endogenous peroxidase activity. The following primary antibodies: Rabbit anti-HIF1α (1:100; catalog no. sc-10790; Santa Cruz Biotechnology, Inc., Dallas; Texas; U.S.A) and rabbit anti-p-S6 (1:100; catalog no. 4858; Cell Signaling Technology, Inc.) were applied overnight at 4°C. Subsequently, sections were washed three times with TBS and then incubated with SignalStain^® Boost Immunohistochemistry Detection reagent (1:50; catalog no. 8114; Cell Signaling Technology, Inc.) for 1 h at room temperature. Sections were then incubated with 3,3′-diaminobenzidine (ZSGB-BIO, Beijing, China). Finally, the sections were dehydrated, mounted and examined under a light microscope (Leica Microsystems GmbH). Positive cells were counted using Image-Pro Plus software verison 7.0 (Media Cybernetics, Inc., Rockville, MD, USA).