Table 2.

Determine antibody requirements

Monoclonal (mAb) vs. polyclonal antibody (pAb)	mAb - single epitope, single isotype, unlimited supply of identical reagent, identifiable (usually) by clone name. pAb - multiple epitopes, better for some techniques as recognizing a range of different epitopes can increase the number of suitable technical applications and enhance signals by enabling more antibodies to bind the same antigen molecule and by forming large precipitating lattices. Conversely, there is more risk of cross-reactive epitopes if the immunogen shares identity with other proteins because a polyclonal antibody will recognize a range of different epitopes. Batch variability, caused by limited quantity and differences in the immune response during subsequent production in another animal, requires additional validation. Sometimes it can be hard to conclusively identify reagents used in the literature or distinguish them among those offered by multiple suppliers.
Isotype	Different isotypes may be useful for experiments using multiple antibodies e.g. enabling detection using isotype specific secondary reagents. In both in vitro and in vivo studies, ‘isotype-dependent’ or ‘isotype specific’ antibody functionality may be important.
Host species	As above it can help to have different species for multiple labeling. Also the use of same species antibodies and tissues needs additional strategies to avoid secondary antibodies detecting endogenous immunoglobulins, this can be avoided if the antibody is raised in a different species.
Define intended technical applications	Consider the intended use in technical applications such as IHC, IHC-P, ICC, IF, FCM, ChIP, WB, ELISA, IP. Abs that recognize linear epitopes (anti-peptide Abs) tend to work well for WB and IHC-P. Abs recognizing native epitopes tend to work well for IP, ELISA and FCM (Abs raised against native proteins, cDNA immunization, cell-based immunogens). While antibodies against extracellular epitopes are commonly used for FCM and for antibody therapeutics, FCM is also increasingly used for antibodies recognizing both intracellular and intranuclear epitopes.
Identify individual antibodies	Refine the list of antibodies using their unique identifiers to remove duplicates such as the same antibody being available from different suppliers. Use the clone name to identify monoclonal antibodies. For polyclonal antibodies, bear in mind that those with an identical host species, immunogen and/or images in validation data may be the same reagent.

Immunohistochemistry (IHC), Immunohistochemistry on formalin fixed and paraffin embedded tissues (IHC-P), immunocytochemistry (ICC), Immunofluorescence (IF), flow cytometry (FCM), chromatin immunoprecipitation (ChIP), Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), immunoprecipitation (IP).