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Finally got the cytof.uk user group website off the ground - thanks to those of you who have been waiting patiently.

Last week, we talked about classical IHC, and how a new technique could revolutionize this application like we have never seen it before. The technique, named multiplexed ion beam imaging (MIBI), has been used recently to analyze formalin-fixed, paraffin-embedded human breast tumor tissue sections stained with ten labels simultaneously. As we explained before, a major limitation of IHC is the low multiplexing capability. Ten parameters sounds pretty amazing already, and the developers are mentioning even more, imagine that! So how does it work? The principle is similar to mass cytometry, for those familiar with CyTOF® and metal conjugated antibodies. Diagram illustrating how mass spectrometry metal conjugated antibody detection works. CyTOF® and MIBI work in a similar way. Image taken from the Blau Lab, Stanford (http://web.stanford.edu/group/blau/flow_cytometry.html). In the particular case of MIBI, metal conjugated antibodies are also detected using a mass spectrometer. The long distance between the metal peaks makes this method free of spill-over signal. After collection and quantification of the signals, dedicated software converts them into images that are easier to analyze. The image of the sample surface is digitalized (rasterized) after being hit with an oxygen primary ion beam that makes the metal conjugated antibodies emit secondary ions that will be detected by the mass spectrometer. MIBI workflow. Image based on a figure from a paper published by Dr. Garry Nolan, Stanford University. It may not take too long before we see a revolution in tissue stained for IHC. Thus far, the images produced by MIBI are beautiful and no doubt they will be very useful too. However, there are some factors that need to be considered in the near future, and before MIBI becomes a widespread application. As with any new technique, there are some pros and cons to be evaluated, when compared to traditional IHC. Advantages of "MIBI" IHC over "conventional" IHC: The use of many markers simultaneously. This will allow for more powerful research and diagnostic applications. There is also lower, if any, background or autofluorescence involved. Very clean images can be generated as a result of the absence of overlap in the signals. Limitations: It requires complex, expensive instruments and analysis tools. In addition, as more markers are added, longer acquisition times are required. This may improve in the future. Despite the disadvantages, there is no doubt that the boundaries of science will be pushed forward once again with the development of MIBI IHC. These two posts were inspired by the work of Dr. Garry Nolan and his team, which was recently recently published in Nature Medicine. Click here to read it. If you have any comments, please let us know: [email protected] Contributed by Miguel Tam, PhD.

Many of today's research techniques are built upon incremental improvements of technology over time. Classical tools such as flow cytometry and immunohistochemistry have evolved along with the instrumentation and reagents required to perform them. In the case of flow cytometry, the instruments and the dyes used have evolved tremendously in the last few years. The discovery of new dyes, such as the Brilliant Violet™ line, and the engineering of new instruments have propelled the field to completely new heights. In the case of immunohistochemistry, if you type that term into PubMed, you will retrieve over half a milion papers. The first paper dates back to 1962. But, what exactly is immunohistochemistry (IHC)? IHC combines anatomical, immunological and biochemical techniques to identify discrete tissue components. Specific antibodies tagged with a detectable label will bind target antigens, and a detection system and instrument, a microscope, is used to visualize the reaction. Thus, IHC is tightly bound to the development of microscopy. Microscopes are among the oldest instruments for biological research. In the late 17th century, Anton van Leeuwenhoek (1632-1723), a Dutch draper and scientist, was the first man to make and use a microscope for the analysis of microscopic cells and particles. By the way, have you seen the "origami" microscope? This is a microscope that is built out of paper. Just imagine the implications on costs, usability, etc. of this device. Read more about it here. Illustration by Foldscope team. There can be some confusion regarding the IHC terminology. Here we will define classic IHC as being performed in preserved tissue, involving sectioning and detection of the antigens with antibodies. The most common detection system is the chromogenic deposition of a substrate near the antigen, exploiting the activity of the Horseradish Peroxidase enzyme (HRP). However, with the development of fluorescent microscopes, visualization can be done using fluorescent, conjugated antibodies. In fact, the first IHC experiments are credited to Dr. Albert Coons, who developed the first methods to conjugate antibodies to fluorescent tags, hence doing “fluorescent” (instead of chromogenic) IHC. His first paper related to IHC was published in 1942. You can read it here. Albert H. Coons Over the last 6 or 7 decades, the principles and applications of IHC have remained pretty much the same since its introduction. A strong limitation of IHC is the number of parameters that can be detected. Unlike flow cytometry, where a clear distinction can be made on more than 10 parameters, the capacity of multiplexing IHC is very limited. However, this is going to change in the future, and there is potential to multiplex those 10 parameters, or even more, in the "new" IHC. How does it work? Come back next week to find out! Click on the picture to watch a video about how IHC works. Chromogenic stain for CD10 in Kidney. Fluorescent stain for actin in the smooth muscle of the skin. Please leave us your comments: [email protected] Contributed by Miguel Tam, PhD.