Western Blot: C2C12 cells were infected with adenovirus GFP control (Ad-GFP) or HA-Irisin (Ad-Irisin) and cell lysates were subjected to western blot analysis by staining with our anti-Irisin antibody (500 ng/ml). Both the 18 and 21 kDa bands are irisin, resulting from multiple FNDC5-derived fragments in different cell lines and tissues due to alternative splicing and post-translation modifications. Antigen neutralization studies and FNDC5 knockout studies in HCC cells has been done to validate the protein bands detected by western blot in collaboration with Professor Ming-Hong Tai from National Sun Yat-sen University in Taiwan. The 25 kDa band shows the HA-tagged irisin.
IHC: Paraffin-embedded mouse tissues were stained with anti-Irisin (1 ug/ml) by immunohistochemical analysis. The paraffin-embedded mice tissues were sectioned into 3-mum slices and mounted on poly (l-lysine)-coated slides. After deparaffinization, the slides were blocked with 3% hydrogen peroxide for 10 min and subjected to antigen retrieval with microwave in 10 mM citrate buffer for 15 min. The slides were incubated with irisin antibodies (1 mug/ml) at 4 degree C After wash with PBS, the sections were incubated with horseradish peroxidase/Fab polymer conjugate (polymer detection system; Zymed Laboratories, South San Francisco, CA) for 30 min and detected using diaminobenzidine (1:20 dilution; Zymed Laboratories).
IF: Immunofluorescence analysis of FNDC5 expression in cardiomyocytes and endothelial cells. Cultured cells were fixed, permeabilized, then stained with anti-Irisin (1 ug/ml) by immunofluorescence analysis. After fixation, the cells were permeabilized then stained with anti-Irisin (1 mug/ml) at room temperature for 1 h followed by repeated washes with PBS. Then, the slides were incubated with fluorescein 5-isothiocyanate (FITC)-labeled secondary antibody (1?500 dilution) for 30 min at room temperature. After mounting in anti-Fade media, the slides were visualized under a fluorescence microscope.
ELISA: Data demonstrates detection of irisin after gene delivery following ELISA protocol
ELISA Kit
CLIA Kit
Assay Kit
Antibodies
Polyclonal Antibody
Secondary Antibody
Antigen
Biochemical
cDNA Clone
siRNA
Peptide
Rec./Purified Protein
Custom ELISA Kit
Custom Protein
Custom Antibody
Antibody Matched Pairs
Antibody Peptide Pairs
Phospho Antibodies
Products by Disease
Products by Pathway
Products by Tissue