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I have seen biologists use mouse grown primary antibodies in mouse tissue, and they've told me that if the blood is perfused well then there is no problem with this method. How does the secondary antibody (anti mouse) not stick everywhere on all the mouse antigens in the tissue?

Clarification: I understand that the secondary antibody is very specific to all mouse "class" primary antibodies. Can I ask then why using a secondary mouse antibody does not recognize background antibodies in the tissue, the ones that the mouse produced for itself naturally before I added any of my primary antibody? Are the existing primary antibody levels already present in the mouse much lower than the levels of the primary antibody I am adding?

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The reason why the secondary antibody only recognizes the primary antibody is relatively simple: It is directed against the Fc-part of the primary antibody. As this part is species specific (but constant between all antibodies from one species, hence the name constant) a secondary anti-mouse antibody is directed against all mouse antibodies.

The general principle can be seen in the figure (although this is from immunolabelling, the principle stays the same, Image from the Wikipedia):

enter image description here

The anti-mouse secondary does recognize the antibodies which are present in the tissue. These are usually present at low levels (unless you work with immune tissues as spleens) and only cause a relatively low background. If it causes a problem, you can treat your sample before you add the primary with an Fc block which blocks the antibodies present in the tissue so that the secondary antibody will not recognize anymore later.

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  • $\begingroup$ I think we essentially said the same. $\endgroup$ – Nandor Poka Mar 20 '15 at 14:16
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Antibodies are very specific on what they recognize (this is called epitope that is the small part of the antigen that is recognized by a specific antibody), and secondary antibodies are made to attach to a specific part of the primary antibody - you can achieve this with monoclonal ABs. Antigens can be recognized by many antibodies (because they can have many epitopes for different antibodies), but usually a single antibody recognizes only a few or more likely a single antigen at max (since the specific epitope it attaches to is only present on a few or a single antigen(s)).

The wiki page for antibodies has plenty of detail on how ABs work.

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