I'm interested in visualizing CD4+ cells to better understand how CD4+ cells are recruited to the thalamus following brain injury. Most papers I've come across use flow cytometry to count the number of CD4+ cells in tissue. However, as a lab we're very inexperienced in flow cytometry, and would prefer to use IHC wherever possible. I have encountered a few papers that use IHC to detect CD4, but relative to the number that use FACs, they are few and far in between. Why is this particular cell marker better captured using FACs rather than IHC, and, conversely, what are the disadvantages to using IHC to detect CD4+ cells?
Dear Deanna
The answer to your question will depend on whether you plan to study this population (CD4 cells) in blood or tissue (eg. brain). If it is blood, then flow cytometry is arguably the best way to enumerate and characterise this cell subtype, study co-expression of other relevant cell surface molecules (eg. adhesion molecules, cytokine/chemokine receptors, co-stimulatory molecules and numerous others). On the other hand if you are going to study a tissue, then you will need to go for immunocytochemistry or immunohistochemistry using fluorescent or conventional dyes. For research, confocal microscopy would be great to get more detailed images of surface/intracytoplasmic details. Hope that helps. Please let me know if you need anymore info.
Good luck
Siva
Muttuswamy Sivakumaran Thanks for your reply! We are looking at brain tissue. However, my understanding from the general literature is that the antibodies for these immune cell markers are not as compatible with IHC as they are with FACs. I would prefer using IHC because it's easier for us and it's in situ, but it looks like in tissue or not, people generally use FACs for markers like CD4. Is that correct?
Not really. Flow Cytometry is really for liquid samples like blood, CSF, pleural fluid, ascitic fluid etc. For tissues like brain, you need IHC. What you are doing is correct and you should carry on. One thing to remember- please make sure that you purchase the right type of antibody for your purpose. Some are optimised for IHC while others are meant for FC.
Good luck
Siva
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