If you are a researcher who is using fluorescent staining on your cells and tissue sections, you should seriously consider using fluorescent tyramides. Why? Well, because they can have exquisite sensitivity, are easy to use, versatile and there is an increasing range of fluorophores in the pipeline. As they rely on a peroxidase-catalyzed visualization process, they are easily integrated into established peroxidase detection protocols. Furthermore, they have the advantage of working at more dilute antibody concentrations and with improved sensitivity. We have used them successfully for the detection of protein (immunohistochemistry and immunofluorescence) and mRNA (‘RNAscope’).

What would we do without fluorescent tyramides?

We run a multi-user, multi-discipline core research within the University of Edinburgh (SuRF). Tyramide reagents are regularly used within the facility—in fact, they are a critical reagent due to their ability to ‘standardize’ multi and same-species colocalization and multiplexing experiments (protein and mRNA).

No cross reaction

A unique property of tyramides was discovered by Zsuzsanna Tóth and Éva Mezey and reported in the Journal of Histochemistry and Cytochemistry in 2007 (click here for the article). Toth and Mazey demonstrated that tyramides will remain bound to the target of interest following a mild heat-induced epitope retrieval step that removes previously bound antibodies from the cell or tissue section. This eliminates the risk of antibody reagents cross-reacting with each other in multiplex colocalization studies and giving false positive colocalization results.

Same species? That’s okay

In principle, this means that antibodies that have been raised in the same species can easily be used in colocalization experiments and traditional HRP conjugated secondaries, streptavidin HRP, and polymers can be utilized to push the sensitivity. A further advantage in our hands is that we are able to automate this staining procedure using immunostaining robots equipped with onboard antigen retrieval capability- a considerable advantage if multiplex immunofluorescent staining is to be delivered to multiple users as a service.

The possibilities are endless

In our multi-user histology facility, it was extremely difficult to develop multiplex protocols for our researchers using traditional combinations of direct, indirect, or streptavidin-based detections. Such an approach would have necessitated stocking a large range of antibodies and detection systems to accommodate the large number of possible permutations we needed to address. We have a large number of users who are interested in using a diverse range of tissue from a diverse range of species and, of course, the primary antibodies which each person wants to use also come from another diverse range of species- the possibilities are endless!

Factors that needed to be considered for our users were;

  • Choice of secondary antibody. Was it suitably cross-absorbed so that it was not going to cross-react with the target tissue or other antibodies in the detection system?
  • Was the detection system of suitable sensitivity to detect low-abundance target proteins?
  • What controls do we need to perform to guarantee that “co-localization” was real and not an artifact of inappropriate reagents cross-reacting and giving false co-localization?

Using standardized tyramide-based detections and confocal microscopes with multi-tracking capability we were able to standardize protocols and routinely visualize four to five colors.

How many?!!

By employing spectral unmixing when imaging, and with the availability of new tyramide reagents, it should easily be possible to push this number even further. In a recent publication, Gerdes and co-workers used iterative staining following the chemical inactivation of fluorescent Cy dyes and sequential imaging with accurate registration of fiducial points. This enabled them to detect 61 different protein epitopes in a single FFPE section! Sixty-one!

The future is bright

The use of peroxidase-labeled primary antibodies followed by fluorescently labeled tyramides in this type of approach could perhaps lead to even greater sensitivity and allow the visualization of proteins expressed below the detection limit of direct fluorescent detections. Watch this space!

Want to know more about histology? Visit the Bitesize Bio Histology Hub for tips and tricks for all your histology experiments.

References

Tóth and Mezey (2007) Simultaneous visualization of multiple antigens with tyramide signal amplification using antibodies from the same species. J Histochem Cytochem 55: 545-554.

Gerdes et al., (2013) Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue. PNAS 110: 11982-11987.

This article was co-written by Mike Millar and Shelia Macpherson- two ninjas of histology from the SuRF Unit!

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