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Our Abpromise guarantee covers the use of ab9324 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use at an assay dependent concentration.|
|Neutralising||Use at an assay dependent concentration. To yield one-half maximal inhibition [ND50] of the biological activity of hIL-6 (0.5 ng/ml), a concentration of 19.0-23.0 ug/ml of this antibody is required.|
|Sandwich ELISA||Use at an assay dependent concentration. In a sandwich ELISA (assuming 100µl/well), a concentration of 4.0-8.0 µg/ml of this antibody will detect at least 500 pg/ml of recombinant human IL-6 when used with Rabbit polyclonal to IL6 (Biotin) (ab84251) as the detection antibody at a concentration of approximately 0.5-1.0 µg/ml.|
|IHC-P||Use a concentration of 0.125 µg/ml.|
|WB||Use a concentration of 0.2 - 0.4 µg/ml. Used in conjunction with compatible secondary reagents, the detection limit for recombinant human IL-6 is 0.5-1.0 ng/lane, under reducing or non-reducing conditions.|
IHC image of IL6 staining in human spleen formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab9324, 1in250 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
This antibody was raised against an immunogen that is predicted to recognize the glycosylated form of IL6 as well as the IL6delta4 splice variant. The predicted molecular weights are 25 kDa and 17 kDa respectively. The band observed at 50 kDa may represent multimers of IL6 as reported in the literature.
Immunohistochemical analysis of PFA-fixed frozen murine neural tissue sections, labelling IL6 with ab9324 at a dilution of 1/1000 incubated for 24 hours at 4°C in 0.1M PBST with 10% donkey serum. Permeabilization was with 0.1M PBS and 3% Triton X. Blocking was with 10% serum incubated for 1 hour at 24°C. Secondary was a mouse monoclonal Alexa Fluor® 568 conjugate at 1/1000.
ab9324 staining IL6 in human cervical squamous cell carcinoma tissue section by Immunohistochemistry (Formalin/PFA fixed paraffin-embedded sections). Tissue underwent heat mediated antigen retrieval in sodium citrate buffer (pH 6.0). The primary antibody was used at 0.125 ug/ml and incubated with sample at 4°C overnight. A HRP-labeled polymer detection system was used with a DAB chromogen.
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