I would like to detect a phosphorylated protein by immunofluorescence in skeletal muscle sections. Is there any adaptation of the protocol specific for phosphorylated proteins, use of phosphatase inhibitor or something else? A recommended protocol to use?
If you looking at a known phosphoprotein of skeletal muscle there is likely phosphospecific antibodies to detect the protein and then just use a fluorescent secondary antibody.
if I understand your question correctly, u have experience with immunofluorescence with tissue sections and would like to do phospho-antigen staining for the first time.
I do not think there are any special general protocols or precautions for phospho-staining and the success and staining protocol will depend completely on how good your antibodies are. Some tissies could be ex-vivo treated with phosphatase inhibitors, but this will not be helpful if you are interested in the in-vivo situation.
https://www.bd.com/resource.aspx?IDX=17718
If you have the right antibodies, go ahead with standard protocols with good controls ( like co-incubation with phospho/non-phospho-peptide- immunogens sold by many companies, phosphatase treated sections etc.). If you are using a new uncharacterized antibody, it will be easier to study its specificity (target and phospho-specificity) in cell lines before using it in tissue sections. Good Luck.
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