Using standard off-the-shelf antibodies and imaging instruments, most researchers can achieve 3-5 plex imaging in their lab using fluorescent detection. The challenge starts when a researcher tries to go above five as either it requires the use of a technique like imaging mass cytometry, DIY approaches such urea-based stripping or proprietary techniques. The challenge with imaging mass cytometry is that is requires a highly expensive instrument as well as metal-conjugated antibodies and a lot of consumables (e.g. argon). This makes the cost per sample of imaging mass cytometry relatively high wherein the cost of materials alone can be as much as $800 per 1 mm2 image with a dozen markers. Imaging mass cytometry is therefore recommended for applications where a tissue is precious and you NEED to get 15 plus markers from a single slide with minimum background noise as some of your proteins might have low expression that would blend into the background with fluorescent imaging.
Over the last few years with the explosion of immuno-oncology therapeutic research and the desire to study multiple immune cell sub-types from a single slide, there have been a wide range of multiplexing protocols described in the literature for fluorescent imaging. However, the exact approach that you take to multiplexing in your lab depends on a balance between cost, throughput and validation. We find that a lot of times while an imaging approach is technically feasible such as imaging a whole mouse brain at 40X using light sheet microscopy, there are typically better ways to address the same research question while minimizing complexity and cost. For example, using two serial sections with five labels each will allow you to achieve ten-plex imaging appropriate for most research questions without requiring expensive and troublesome commercial approaches.
If you are interested in learning more about multiplexing or applying it to your next research project, reach out to us today to discuss our multiplex and advanced imaging capabilities.