Question

For an immunocytochemical experiment in which you labeled tissue with two primary antibodies (one made in rabbit, one made in mouse) you want to visualize the primary antibodies individually with secondary antibodies labeled with fluorophores. In the freezer you find aliquots of goat-anti-rabbit-CY2 and goat-anti-mouse-DL488. Is it a good idea to use these secondary antibodies or not? Provide the reason for your assessment.

Answer 1

Ans) It is given that the tissue is labelled with two primary antibodies i.e one made in rabbit and other made in mouse. In order to detect these primary antibodies with the help of secondary antibodies linked to fluorescent dye, it should be noted that the secondary antibodies should be used such that they should be prepared from animals except which is found in primary antibody (For example,if the primary antibody is prepared from dog,then the secondary antibody can be prepared from any animal except dog).

The tissue which is labelled with primary antibody from rabbit,the secondary antibody to be used should be goat-anti-mouse-DL488 and the tissue which is labelled with primary antibody from mouse,the secondary antibody to be used should be goat-anti-rabbit-CY2.

If the primary antibody used is from mouse and if we use secondary antibody as anti-mouse,this will lead to non-specific binding and gives high background in the experiment which is not good for the accuracy of the experiment.