Biotin Anti-Fibronectin 抗体 (ab6584)

Thank you for your enquiry about our fibronectin antibodies. The antibody ab6584 is biotin conjugated and has been successfully used in IF (ICC) by a user of the antibody who left a review which can be accessed via the online datasheet. The reviewer did not leave many details about the cells or protocol used so you may wish to contact him by clicking on his name on the sheet. As the antibody is biotin conjugated you will need to add an extra-avidin fluorescence conjugate (or avidin), so that the avidin part will bind to the biotin molecules (so you won't need a secondary antibody per se). You can purchase extra-avidin FITC for example and incubate it 1-3 hours with the cells; we currently have no information about the use of ab6584 in IF on sections. We have a similar anti fibronectin antibody unconjugated, ab299, but it has not been tested in IF/ICC or IHC, therefore we do not know if it will work in these applications. Please do not hesitate to contact me if you would like some protocol help with our antibodies or any further information,

For testing 1ug of antigen, the minimum of antibody used is 1:8000. The maximum is 1:4000. We don't have other data to show the sensitivity. If you have any additional questions, please contact us again.

1:4000 to 1:8000 dilution can detect 1.0ug of Fibronectin. If you have any additional questions, please contact us again.

I have not found reports of staining of fibronectin in the cytoplasm of plasma cells, however it is known that plasma cells adhere to fibronectin (the fibronectin inducing not only the adherence and spread of plasma cells but also their migration). I therefore think that the staining you are observing is not specific (especially using normal rabbit serum as blocking agent) and would like to suggest the following changes to your protocol: -what type of IHC are you testing, is it paraffin embedded tissue? If so you need to consider antigen retrieval methods (heat mediated, enzymatic) - you did not mention quenching the endogenous peroxidases prior to start the staining protocol, please make sure you do as the DAB step will give high background if you have not incubated the tissue in H2O2 at the beginning of the protocol to quench endogenous peroxidase -block the tissue in normal goat/horse serum (not rabbit, as the primary antibody will bind to the serum, giving high background/non specific staining) -try decreasing the concentration of the primary antibody and changing the incubation time, temperature and the presence of triton in the dilution buffer -Incubation in ABC could be reduced to 1/2h I hope these recommendations will help you, please do not hesitate to contact me again if you still have problems,

Thank you for your enquiry. We would like to confirm that the concentration is 2.5mg/mL, so 40uL will give 100ug of antibody.

This antibody does not require antigen retrieval but it may increase staining. We normally recommend the pressure cooker method of antigen retrieval.

450 kDa non-reduced 225 kDa fully reduced

This antibody will cross-ract with mouse fibronectin using formalin fixed paraffin embedded tissues.

Yes, this antibody is available in an unconjugated form, ab299

The extracellular matrix proteins are well conserved, particularly between mammals, so human will cross-react with pig.