Main Users and Applications
- Pathology.
- Oncology for Cancer study.
- Animal research.
- Pharmaceutical and biotech company RnD.
Methodology
- Mostly focused on abnormal gene expression, which can lead to various diseases.
- The method uses antigen-antibody reactions, where users detect the presence of abnormal proteins which are the result of the expression of “bad genes”. The targeted protein is the key indicator of such diseases.
- Most of the time, IHC is used to detect proteins related to tumors and cancer. The protein generated from the “bad gene” is called the “Oncogene”. The presence of any of these abnormal proteins help identify if the patient is suffering from cancer; what type of cancer they have; how many and which oncogenes are expressed; we can also deduce the prognosis of the patient. It is also important for the clinician to decide what type of therapy strategy should be used; and if targeted drug or Immuno-therapy can be used.
Application Details
- There are three types of IHC, depending on where the antigen is located: the nucleus, membrane or cytoplasm.
- Generally, a specific antibody (commercially available) is used to tag the targeted protein (Antigen), and a brown color is developed by an enzymatic reaction.
- The density and darkness of the brown color in the cell shows how much of the protein is present.
- Laboratories either do manual scoring by naked eye under a microscope or they will use software to measure the intensity (Gray levels) and the area of the brown color to reach their result.
- There are limitations on accuracy of the method, both in terms of experiment limitations (non-specific binding) and artifacts in result reading. However, the method is commonly adopted as the first line diagnosis approach because of the maturity of the technology and relatively low cost of the process.
Microscope Requirements – What to Look For
- Ergonomics are very important for users.
- Color temperature is important, as accurate colors are needed in order to better see the brown shades.
- The important objectives needed for IHC measurement are usually 20x and 40x.
- Resolution has a lower requirement than what is needed for other applications, such as H&E. However, users will need very good homogeneity of their field of view (even illumination).
- Contrast is considered to be much more important than resolution.
- A convenient way to take photos is almost always a must, as sample images are needed for reports.
- A Gray level measurement software with an easy-to-use, yet accurate color segmentation feature is needed by some users.
- A decent digital camera is expected.
Recommended Specifications
- Both PA53 and PA43 are great fits for this application. The PA53 has some advantages between the two because of the complete optical path with more even illumination.
- As the microscope will likely be heavily used in the laboratory, robustness and sturdiness becomes a prime consideration. This makes our heavy and stable PA43 and PA53 the best options for the applications.
- Plan UC 20x and 40x objectives are the most cost-efficient options.
- Moticam S-series is a great option for IHC, providing high definition at good framerate.
- If IHC measurement software is needed, Motic can easily integrate with many dedicated software solution providers. For example, the Motic Analysis Software has perfect integration with Image Pro Plus (IPP) from Media Cybernetics, which is a commonly used software for IHC measurement.
Motic Setup Recommendations and Specifications
- A heavy, very sturdy and robust stand.
- The IL manager allows the microscope to memorize lighting for each objective, minimizing light adjustment time for users when switching between different magnifications. It is extremely helpful for IHC, as frequent changing between the 10x, 20x and 40x objectives is necessary.
- The Motic S-series camera is great for its price-performance ratio.
- The seamless integration of Motic cameras with IPP software is also a bonus in IHC study.
Figure 1. Courtesy to Department of Pathology, Capital medical University
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