I have 3 groups of animal apart from PBS controls where i treat them under different immunosuppressive conditions. when the animals are subjected to partial or no immunosuppressive conditions, i do see the nuclear expression of BCL-6. When the animal is completely immunosuppressed, i do not see nuclear expression of BCL-6 rather than cytoplasmic. Also, the staining is very faint, eventhough i tried different concentrations. Can i know what would be the reason for cytoplasmic exprression and why do i see only faint staining even with antigen retrival?
I have attached some images for reference.
thanks in advance.
Naren
I strongly advise to switch to fluorescence microscopy and add nuclear markers such as DAPI or even a cytoplasmic marker such as B-tubulin. DAB staining has its limitations in terms of signal to noise ratio and while you can add hematoxylin immunostaining it might be difficult to obtain conclusive data. Another way to investigate the nuclear and cytoplasmic presence of the target protein is to perform nuclear and cytoplasmic fractionation and run western blot with the fractions, but since it requires fresh tissue switching to immunofluorescence might be an easier approach.
Anton Lennikov thank you very much. I will give it a try. Any idea why would it be present in the cytoplasm. As far as i understood, if it is present in the cytoplasm, it is marked for degradration or in the process of drgradation? what are your thoughts on it?
@daniil I used BSA and H2O2 in methanol for Fc and peroxidase blocking
Hi Narendran,
BSA block common nonspecific binding, but not an interaction FcR with IgG.
Depending on your primary and secondary antibody sources add a blocking step with serum (goat serum good point to start) before incubation with Ab. It should reduce membrane signal. This step is extremely important since the spleen full of macrophages and other cells expressing FcR.
H2O2 should block peroxidases, methanol should facilitate access to intracellular space.
Anyway, for the first experiment, it is better to check the specificity of staining by introducing negative control without 1st Ab incubation (just the same buffer). As well as to use tissue with know positive control.
If staining will not appear I would check antigen retrieval protocol and wild detergent conditions (Triton X-100, NP-40).
Keep in touch!
Daniil
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