Primary and secondary antibody selection for immunohistochemistry.
Selecting a primary and secondary antibody can present a number of challenges.Ā In order to help immunohistochemistry labs make this selection process easier, presented here are tips from experts in the field.Ā There are a number of considerations and concerns in antibody selection, some of which are covered below.
Primary Antibody Selection
The first consideration is determining ifĀ the primary antibody of interest is reported to bind specifically to the perceived expressed target antigen in the speciesĀ and cell typeĀ you are interested in labeling.Ā For instance, there is a very good antibody made in Rat against mouse CD31, but it only works on mouseĀ endothelial cells.Ā Ā If you wanted to label endothelial cells in rat, you would have to find anotherĀ endothelial cell markerĀ that will bind toĀ ratĀ cells.
Sample preparation should be considered when choosing the appropriate antibody.Ā Ā It is important to know ifĀ the antibody has been reported to work onĀ samplesĀ that haveĀ been fixed and processed the way theĀ samplesĀ being studied haveĀ been.Ā Some antibodies will not work on formalin fixed paraffin embedded (FFPE)Ā samplesĀ and require frozen sectionsĀ that are non-aldehyde fixed in something such asĀ acetone, or a specialĀ non-aldehydeĀ zinc salt fixativeĀ for paraffin processed samples.
Further, attention needs to be paid toĀ whatĀ speciesĀ theĀ antibodiesĀ are made in and what species they are against.Ā Ā Ā It is best that the primary antibody be made in aĀ species different from theĀ target species, even though an antibody made in the same species you are targeting may bind to that antigen in that species(species cross reactivity).Ā The reason this is a problem is that if you are using a mouse antibody on mouse tissue that is not directly conjugated to something, in order to detect the binding, you have to use some kind of secondary reagent which would be made in another species against mouse immunoglobulin; all mouse Ig, not just the primary antibody Ig.Ā There would be binding of endogenous Ig in the mouse tissue unless special blocks are used to block the endogenous mouse tissue Ig.Ā Antibodies made in rabbits or goats are most commonly used on rodent tissue to avoid this issue.Ā That is why rabbit monoclonals are so popular in rodent IHC.
In addition to these considerations,Ā you should know what Ig isotype the primary antibody is so you can best match it to that specific secondary Ig isotype.
Secondary Antibody Selection
The most important considerations for choosing a secondary antibody are as follows:
Secondary antibodies should be made in a different species against the species the antibody is made in.Ā For example, if you have an antibody made in aĀ rabbit against mouse antigen, you should choose a secondary that is made in aĀ species such asĀ goat against rabbit so it will bindĀ to the rabbit the primary was made in.Ā If the secondary link is not conjugated to a visualization label such as FITC or enzymes such as horse radish peroxidase (HRP) and alkaline phosphatase (AP), then you need to use a tertiary detection reagent such as labeled polymer with one of these attached to it.Ā That tertiary detection reagent should be made in a species different from the species the secondary is made in.Ā For instance, some very common detection systems made for rabbit and mouse antibodies for human samples use a post primary secondary link Ā which is rabbit anti mouse IgG to label mouse antibodies, followed by goat anti rabbit labeled polymer HRP.Ā To use these detection reagents with rabbit antibodies on rodent samples you can just leave out the post primary rabbit anti mouse reagent and go directly to the goat anti rabbit labeled polymer which will bind directly to the rabbit primary antibody.Ā If you used the rabbit anti mouse post primary reagent on mouse tissue, you would get nonspecific binding of all mouse IgG.
Also,Ā it is best to try and match the secondary Ig isotype to what Ig isotype the primary antibody is.Ā Most antibodies are IgG but occasionally you run into one like CD15 that is IgM and not IgG. So if a secondary, even if you matched the species was IgG instead of IgM it would not work very well.
