Hello,
We are trying to stain actin and tubulin with BACMAM. We have tried different concentrations and incubation times, however each time we get the same type of image.
The cell is stained but there are no lines for the actin and tubulin instead the cells are comprised of dots and there are some very bright larger dots (10-20 per cell) like they might be BACMAM particles not ingested by the nucleus.
Has anyone experienced this? Could it be a bad BACMAM batch? Perhpas the concentration is still too high? The cells have previously been studied with BACMAM without issue.
Thank you.
Dear Lara,
BacMam is technically a virus and it should express tagged version of Actin eGFP and Tubulin eGFP for instance. The cause for the dots to appear cannot be BacMam particles not being imported in the nucleus. They are not stained and need to be in the nucleus to produce Actin and Tubulin. If you see dots and mislocalization it can be two things:
1) You are transducing with too much virus. You are only detecting the brightest (and possibly mislocalised proteins). Try to use less virus and a better microscope.
2) This is more likely in my opinion. Your target cells are not transduced, and you are imaging autofluorescence dots instead of actin and tubulin.
Is your cell line amongst the ones that can be transduced with BacMam? If so option 1 is most likely. Otherwise, it might be well 2.
Hope it helps
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