Using immunofluorescence (IF), I am having quite the problem with the appearance of what appears to be non-specific staining of my primary antibody (mouse IgG1), which is directed towards a nuclear, chromatin associated factor. This non-specific staining appears in the periplasm/cytoplasm only 6-12 hours after infection of these cells with a DNA virus, and occurs only in infected cells, not in surrounding uninfected cells. It is also worth mentioning that the non-specific peri/cytoplasmic staining is vastly more intense than normal staining in uninfected cells. I am claiming that this is non-specific recognition of my primary antibody due to the fact that in my secondary only IFs I get no signal, however using an isotype control (anti-FLAG or anti-GFP) I get the exact same staining pattern in infected cells as when I use my specific antibody. It also doesn't appear to be due to my fixation conditions as I see this with both PFA and Methanol fixation methods. Any help or comments with regards to what may be causing this sort of anomaly and how to get rid of it would be GREATLY appreciated. I have posted below my general IFA protocol and have attached images of staining in both uninfected and infected cells.
Transfer coverslips to a new 6 well dish containing PBS
Rinse coverslips 2x using RT PBS
Fix 15 min @ RT using 4%PFA/ddH20 pH7.4
Incubate coverslips 3x5min in ice cold 0.1M Glycine/PBS
Incubate 10min on ice using 0.25% TritonX100/PBS
Wash 3x5min using ice cold 0.2% Tween20/PBS
Incubate on rocker 1hr @ RT using Blocking Buffer (PBS containing 5%BSA & 0.2% Tween20)
Incubate coverslips in a humid chamber face down on a strip of parafilm containing a 50ul drop of antibody diluted in Blocking Buffer for 1hr @ 37 degrees
Wash 3x5min using cold Wash Buffer (PBS containing 0.2% Tween20)
Incubate in humid chamber as before for 1hr @ 37 degrees using secondary antibody diluted 1:1000 in Blocking Buffer
Wash 3x5min using cold Wash Buffer (PBS containing 0.2% Tween20)
Rinse 2x using ice cold PBS
Mount using Slowfade containing DAPI
Uninfected cell staining.jpg (14.58K)
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Infected cell staining.jpg (17.57K)
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