Peroxidase conjugated goat anti mouse igg

Soleus muscle and C2C12 myotubes protein extraction were performed using Ripa Buffer and Protease/Phosphatase Inhibitor Cocktail (Roche Applied Science, Indianapolis, IA, USA) and protein quantification using the Bradford method. Protein samples were then subjected to 10–15% SDS-PAGE, transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA), blocked with 5% skim milk at room temperature for 2 h, then incubated overnight at 4 °C with primary antibodies of Slow Skeletal Myosin Heavy chain/MyHC-I (1:1000, ab11083, Abcam, Cambridge, UK), Fast Skeletal Myosin Heavy chain/MyHC-II (1:1000, ab91506, Abcam, Cambridge, UK), and Vascular Endothelial Growth Factor (1:1000, ab1316, Abcam, Cambridge, UK). Membranes were washed in TBST three times and incubated with relative secondary antibodies (Peroxidase-Conjugated Goat anti-Rabbit IgG; Peroxidase-Conjugated Goat anti-Mouse IgG, ZSGB-Bio, Wuhan, China) for 1 h at room temperature. Subsequently, the membranes were washed in TBST three times and protein bands were visualized with enhanced chemiluminescent agents using Fusion FX (Vilber Lourmat, Marne La Vallée, France) and quantified with Gel-Pro Analyzer (Media Cybernetics, Rockville, USA). β-actin (1:1000, sc-47778, SantaCruz, Dallas, TX, USA) or β-tubulin (1:1000, 2128s, Cell Signaling, Beverly, MA, USA) were used as loading controls.

The protein samples were extracted from the small intestines and IEC-6 cells by RIPA buffer added with a protease/phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA).
The protein samples were electrophoretically separated using 12–15% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h with 5% dried skimmed milk at room temperature, incubated overnight at 4 °C with primary antibodies listed in Table 2, and then incubated for 1 h with the corresponding secondary antibodies (peroxidase-conjugated goat anti-rabbit igg; peroxidase-conjugated goat anti-mouse IgG, ZSGB-Bio, Wuhan, China) at room temperature. Finally, the proteins were visualized using a chemoluminescence system (Fusion, Paris, France) with Millipore Immobilon ECL substrate (Millipore, Burlington, MA, USA). Densitometry analysis was conducted by Image J V1.8.0.112 software.

Specimens were fixed in 10% formalin solution and embedded in paraffin wax. 4 μm serial sections were cut from the tissue blocks, deparaffinized in xylene, and hydrated in a series of alcohol (75%, 85%,95%, 100%), followed by antigen retrieval with EDTA. Tissue sections were then incubated with primary antibodies (FYN, TOPK and p-TOPK(Y272)). Subsequently, tissue sections were incubated with secondary antibody (Peroxidase-conjugated goat anti-rabbit Ig, ZB-2301, Zsbio, China; peroxidase-conjugated goat anti-mouse IgG, ZB-2305, Zsbio, China) for 2 h at room temperature, and then stained with DAB kit (ZL1 − 9018, ZSGB-BIO, China). After staining, sections were digitally scanned using the Aperio AT2 scanner (Leica Biosystems, Germany).