I generally flush the rat with saline before harvesting tissues for western blots. I have heard that the blood and other components can interfere with blots. Does anyone else flush the animal before harvest?
Kellie, depends what you want to detect. To find out if plasma or serum have an influence on your data, run samples of them in your gels. If there is an effect, I'd suggest to include controls for sufficient flushing (i.e. probing for a marker from serum or erythrocytes (probably albumin or Hb) in future experiments.
I think the main reason for flushing the animals is that blood cells might contain the protein that you want to detect with the wb. I flushed the animals because I was doing wb for the carbonic anhydrase 2 and this enzyme is quite aboundant in erythrocytes. But i was never abel to completely remove blood from heart and liver by perfusion, thus you may still expact some contamination.
in case of staining e.g., H-E staining it will be more visible and better when you flush the target organ, to find out whether the blood protein could interfer with you data from the kidneys, one may try on animal with flushing and on without, and check the outcome...
Good luck
Depend what you are looking for. And yes blood flushing will change probably the composition of protein pool in your lysate.
So yes you should consider it if you have in the blood of your animal something you do not want to quantify. However... western blot is not cell type specific, meaning you always end up with a mixture of blood, nerve, fat... But it is not the aim of in vivo assay to have these complex tissue mixture?
If you want to be cell type specific, Salamah pointed out the interest to make histology.
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