I am trying stain for two membrane proteins, one cellular and one viral. But lately, i am doubting a little that the double positive staining could be because of background staining of the other antibody across different filter. In a nutshell, all the viral protein is co-localizing with the cellular protein. Although i am happy, i am still skeptical? I am new to immunofluorescence, help from the experts would be much appreciated.
Much thanks,
Naren
Try some control experiments, e.g.:
- Secondary-only incubation (do everything as usual but leave out the primary antibody, just use the antibody buffer instead)
- Staining for the viral protein in non-infected substrate
-...
In general, to help you it would be useful to know a bit more about your experimental setting, e.g. which substrate, which fixation, which detection system, ...
Yes, make sure you have a secondary control where you only stain the sample with the secondary and no primary. This way you can rule out if it is background that you are seeing in your samples. You can also do one antibody stain to each sample to see if the fluorescence is seen in the same location. Make sure you are following the antibody dilutions recommended for your primary and secondaries. I usually start 1:100 primary and 1:500 secondary and get good results.
Thank you for your valuable comments... I will try and do that same @vivaca and @heather..
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