Hello everybody!!! I overexpress an HA-tagged protein in the DG of the hippocampus (mice) by viral infection. Afterwards, i stain the brain slices against the HA tag by the IHC. As expected, I dont get a signal in the uninfected animals. The background signal also seems to be low. However, in the infected animals, DG is pretty damaged (may be due to the viral lethality) and there is a huge background signal!!!! the signal is as strong as the specific signal, which makes it very difficult to quantify. In general the whole slice seems very greenish (488-secondary antibody) and this "signal" even further increases as it gets closer to the infected&damaged site..
Have you ever experienced a similar situation? Can there be a link between the neuronal lethality (I assume the dead neurons are infected) and the increase in the background signal?
Thank you very very much in advance!!!!!
Well, if the proper control: empty virus infected brain tissue has no backgound and damage,then it is very likely that the overexpressed protein is the cause for the neuronal damage that you see. As dead neurons leak out your tagged and probably somewhat degraded target protein, which explains the described background.
Yes, I have seen this before for an important CNS protein. Protein overexpression is often a serious issue in the CNS.
I think the background could be autofluorescence from the dead cells. To confirm this check the signal in other filters for example in red channel (555) and, 647 where you dont expect any signal in general other than at 488. if you see bright signal at other channels too you can confirm that is an unspecific and autofluorescence. I have experienced this autofluorescence in the spinal cord lesion centre where I expected some dead cells. Hope this helps.
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