Hi,
Iam working with a cervical cancer cell line and Iam supposed to do immunofluorescence for MMP's. Iam facing a wierd problem. When I do the negative control with IgG, (Alexa 488) there is a bright fluorescence like the ones that I needed for the result. I have repeated it twice and still I'm getting the same result.
The conditions are 1in 200 dilution overnight as I do for the test samples. When the same IgG is used for immunohistochemistry, the result is good. I do know 488 has some autofluorescence, but what I see is more than the background level. Please help.
jaya
Could you give details about the antibodies you use? Host species, manufacturer.
Process three sample at the same way, but:
1. do not use any abs.
2. use the sec only.
3. use both.
the dilutions will be the same as you plan to use it.
Results:
1. no signal, or weak autofluorescence of cells.
2. no signal, or weak fluorescence of the sec due to unspecific binding.
3. strong signal from the immunocomplex.
Exposition parameters should be the same, and normal range (about max 1 sec. no gain, gamma is about 0.65). Do not use auto setup of exposure.
Are your primary antibodies conjugated, or do you use primary + conjugated secondary?
So I'm presuming that you see a background when you use the Isotype control of the primary antibody, plus the secondary, but you don't see this same background when you incubate your cells in PBS overnight and then add just the secondary. If this is the case, you might need to lower the concentration of the IgG control, and consequently, the primary anitbody. Make sure you are using the same protein concentration for both, and not just the same dilution factor (usualy isotype controls are of a higher protein concentration). If you see a background also when your cells just have the secondary Ab on for 1hr room temperature, then decrease the concentration of the secondary Ab. You mentioned that this problem didn't occur in IHC, but you probably need to adjust the antibody concentrations for ICC. It's very likely that you will need lower amounts of both antibodies.
- Badges
- Science topic