Question

When performing immunocytochemistry experiments, what is the purpose of the blocking serum? If you only had access to normal mouse serum, from what species would your primary and secondary antibodies need to be derived? Is it possible to fluorescently label two different proteins in the same cell using the same immunocytochemistry techniques? If so, what considerations would you need to make for your primary and secondary antibodies?

Is it correct that the primary and secondary antibodies have to be from different species than the mouse serum?

Also, if anyone you are able to attach some scholarly references as well that would be very much appreciated!

Answer 1

Ans) The role of blocking serum in the immunocytochemistry experiments is to avoid non-specific binding between the antigen and antibodies.Usually,serum (1-5%) is used for blocking. It binds to the reactive sites of the non-specific molecules and improves the sensitivity and specificity of the technique.

If mouse serum is used,the primary and secondary antibodies should be derived from other animals except mouse. Usually, anti-mouse antibodies and anti-rabbit antibodies are used. For example,if you are studying mouse serum,the primary antibody should be derived from other animals except mouse (say you can use anti-rabbit antibodies) . Now,if your primary antibody is Anti-rabbit,the secondary antibody should also be derived from other aninals except rabbit (say you can use Anti-Goat antibody derived from the goats).

It is possible to fluorescently label two proteins in the same cell using same immunocytochemistry technique but the chances of cross-reactivity and non-specific binding of Antibodies are high (For example the Antibody number 1 which is raised against Antigen number 1 can bind to Antigen number 2 and vice versa thus resulting in cross-reactivity which can give false results).However,the advantage of this Double immunostaining procedure is to determine the co-localization of the proteins within the same cell which is determined by confocal microscopy also. Double staining can be done by various methods.One of them which can be used is, to use Antibody number 1 for Antigen number 1 , Antigen number 2 is incubated with avidin and We add strepatvidin antibody for Protein or antigen number 2 . This is one of the method which can be used. There are many such methods which are employed.