Anti rabbit igg hrp

Spinal cord tissue transfected by AQP4-RNAi-LV was collected to extract protein, which was used to assess the protein expression of AQP4 following lentiviral injection. Protein samples were boiled for 10 min. The proteins fractionated on 15% SDS-PAGE gels and were transferred to polyvinylidene difluoride membranes (PVDF) and Millipore membranes for 120 min at 75 V by electrophoresis. The membranes were then blocked in 5% non-fat milk for 120 min at room temperature and incubated with primary antibody of AQP4 (rabbit, ab46182, Santa Cruz, 1:200) overnight (16–20 h) at 4°C. After incubation with the primary antibody, the membranes were washed in three changes of buffered saline TBST and incubated for 120 min with secondary antibody (HRP anti-rabbit IgG, ZSGB-BIO, diluted by 1:5000. Finally, membranes were rinsed in TBST 3 times and the immune complexes were visualized with enhanced chemiluminescence (ECL) and captured on Alpha Innotech (Bio-Rad). Band intensities were determined using densitometry (ImageJ), and AQP4 levels were normalized to beta-actin.

Slides with 4–5 μm tissue sections were baked at 60°C, deparaffinized with xylene and rehydrated through a graded series of ethanol solutions (100%, 95%, 85% and 75%). Tissue slides were then treated with microwave to induce epitope retrieval by boiling slides in critic acid solution for 15 min. Protein blocking was performed using blocking buffer (ZSGB-BIO, Cat# GT101510) for 20 min at room temperature. Primary antibodies for anti-ERO1L (Abcam, Cat# ab177156), anti-IRE1α (Abcam, Cat# ab37073), anti-PanCK (Abcam, Cat#ab7752), anti-CD4 (Abcam, Cat#ab133616), anti-CD8 (Abcam, Cat#ab4055), and anti-CD68 (Abcam, Cat#ab213363) were used. The slides were then incubated with secondary antibodies (HRP-anti-rabbit IgG, ZSGB-BIO, Cat# PV-6001; HRP-anti-mouse IgG, ZSGB-BIO, Cat# PV-6002) for 30 min at room temperature. Each slide was evaluated by 3 pathologists. For mIHC analysis of human samples, heat-induced epitope retrieval was performed to remove all the antibodies including primary and secondary antibodies after each cycle of staining. Multiplex immunofluorescence staining was performed using the AlphaTSA® Multiplex IHC Kit (AlphaX, Cat# AXT36100031). Slides were counterstained for nuclei with DAPI (ZSGB-BIO, Cat# ZLI-9957) for 10 min and mounted in mounting medium. Images were scanned and captured using ZEISS AXIOSCAN 7.

The total protein was extracted from C2C12 myotubes and gastrocnemius muscle using cell lysis buffer for western blotting and IP (Beyotime, China) according to the manufacturer’s protocol. The quantification of total protein was measured with a BCA Protein Assay Kit (Beyotime, China) according to the manufacturer’s protocol.
The primary antibodies to Pan-Akt, P-Akt (Ser473), P-Akt (Thr308), mTOR, P-mTOR (Ser2448), S6, P-S6 (Ser240/244), P70S6K, P-P70S6K (Thr389), 4E-BP1, 4E-BP1 (Ser65) and GAPDH were all purchased from Cell Signaling Technology. Anti-rabbit IgG HRP (ZSGB-Bio, China) was used as a secondary antibody (1:10,000). The blots were visualized using SuperSignal West Dura Extended Duration Substrate (Thermo scientific, USA).