Smarter staining: New biocatalytic systems for multiplexing histological staining

Background: Histopathology is the workhorse of many medical laboratories, and can be used for disease
diagnosis, drug development and biological research. Histological staining is an essential tool for this process,
providing valuable contrast and visually identifying microscopic features in samples. Since it is essential that
this process is both robust and repeatable, innovation has been limited and only a very few technologies have
been employed. Chief amongst these technologies are staining process which use horse radish peroxidase
(HRP) to catalyze the oxidation of the chromogen diaminobenzidine (DAB) to give an insoluble brown stain.
When coupled to an antibody recognition system this can provide a map of any individual target on the
biological tissue sample. With the advent of "personal medicine", multiplexing staining would provide an
important advance in histopathology in terms of the amount of information which could be generated from
any one patient sample, a particularly important feature where tissue is limited. At the moment multiplexing
is limited to the use of mouse and rabbit antibodies linked to two different enzyme/chromogen systems: the
HRP/DAB combination; and an orthogonal alkaline phosphatase (AP)/Fast Red combination.

In this project: an innovative approach to multiplexing will be explored which relies upon the development of
new polymer-bound biocatalyst /pro-chromogen substrate combinations, in which biocatalytic turnover of
the pro-chromogen substrate can be used to multiplex staining.