If I want to test a chemoattractant (it can recruit immune cells to tumor) such as MIP-1a, should I do IHC of tumor stained with MIP-1a staining? I want to test whether after treating with a drug, will the chemoattractant increase in the tumor? In this case, I might not care about which cell secrets this chemoattractant.
Can someone answer me? I think usually people do IHC to see immune cells on the tumor, I don't know whether IHC with a chemokine is logical or not
Thank you so much!
You should do two ways: Immunohistochemistry on paraffin-embedded tissues and by ELIZA of tissue lysate
So, yes you can use IHC to detect secreted proteins.... However, I'm not sure if the proteins once secreted outside the cell will be in enough to be detected, much less to detect a chemokine gradient.
Similarly... I doubt that you can use the ELISA to detect a gradient. Also, you cannot use FFPE tissues for ELISA. The only way I can see an ELISA will work is on fresh tissue and you have to isolate the tumor and find a way to normalize your results.
Usually, chemoattractant is usually a functional assay. Which means that ideally, you probably want to isolate the tumor cells with or without treatment, and use a chemotaxis assay. The most common is using a transwell system of some sort that has a membrane to allows cells to migrate through.
If all you are doing is to see if the drug can induce production of the chemokine, yes then IHC is the way to go. But again, for secreted proteins... it's difficult to say if it would be in high enough concentration for the IHC to work.
Now... you can try an organ/tumor lysate ELISA. This is pretty simple. You can do it in different way depending of what your goals are.
1. Excise the tumor, homogenize it, and just simply test the lysate by ELISA.
2. Excise the tumor, homogenize it, incubate it for like 4 hours or so in media, and then test the supernatants by ELISA.
The normalization you can roughly do it by organ/tumor weight. Or you can try to do it by total protein.
The only problem with the organ/tumor method is that you won't be able to say what cell actually is producing the chemokine of interest.
Although, if you know which tumor cells you are looking at, it's better to see if you can find a cell line that is similar and do a cell culture experiment first, instead of going right into animals.
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