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Our Abpromise guarantee covers the use of ab22554 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ELISA||Use at an assay dependent concentration.|
|ICC||Use at an assay dependent concentration. PubMed: 19657392|
|ICC/IF||Use a concentration of 2.5 µg/ml.|
|IHC-P||Use at an assay dependent concentration.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 30 kDa (predicted molecular weight: 26 kDa).|
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) of Mouse lipid staining Adiponectin useing ab22554 (PBS in negative control) at 1:200. ImmunoHistoProbe one step HRP Polymer (ready to use) used. Performed heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0)
ab22554 staining Adiponectin in 3T3-L1 cells (ATCC® CL-173 TM). Increased expression of Adiponectin correlates with adipocyte phenotype, as described in literature. Cells, grown to confluency in DMEM with 10% FBS, were differentiated by stimulation for two days with 0.5 mM 3-isobutyl-1-methylxanthine (ab120840), 0.25uM dexamethasone (ab120743) and 1ug/ml insulin (ab123768), followed by two more days with only insulin. Cells were maintained for an additional three days in growth medium alone. Undifferentiated and differentiated adipocytes were fixed with 100% methanol (5min) at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 1h at room temperature. Staining of the treated cells with ab22554 (2.5µg/ml) and ab6046 at 1µg/ml overnight at +4°C, was followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Rabbit secondary (ab150081) at 2 µg/ml (shown in green) and AlexaFluor®594 Goat anti-Mouse secondary (ab150120) at 2 µg/ml (shown in pseudo colour red). Nuclear DNA was labelled in blue with DAPI. Negative controls: 1– Rabbit primary and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
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