Method Article
Dephosphorylation of epitopes in murine skin sections
https://doi.org/10.21203/rs.2.10179/v1
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posted 30 Jul, 2019
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Dephosphorylation of epitopes is a common way of testing phospho-specificity of antibodies. Whereas it is a regularly used protocol for dephosphorylation in cell lysates, its use in immunohistochemistry has been limited. Here we describe a methodology to dephosphorylate adult murine tissues by using Antarctic Phosphatase, an alkaline phosphatase. This protocol is compatible with subsequent immunohistochemistry, making it suitable for validating phospho-immunoreactivity in tissues.
tissue dephosphorylation
epitope dephosphorylation
alkaline phosphatase
antarctic phosphatase
Xylol
Isopropanol
Ethanol
H2O
Dako Antigen Retrieval Agent pH9 (Cat.No. S2367)
Phosphate Buffered Saline++ (PBS plus 0.2 mM CaCl2, 0.2mM MgCl2)
Antarctic Phosphatase (AP) and AP buffer (50 mM Bis-Tris-Propane-HCl, 1 mM MgCl2, 0.1 mM, ZnCl2 ,pH 6) (NEB, M0289S)
Bovine Serum Albumin (e.g. SIGMA A7906)
Boiling cuvette
Pressure cooker
Fat pen (e.g. Dako pen)
Humidified chamber
Incubator 37°C
Deparaffinization
Deparaffinize the tissue sections from adult murine skin by submerging the sections in the following alcohol row: xylol for 5 minutes, fresh xylol in a different container for 5 minutes, isopropanol for 5 minutes, 96% ethanol for 5 minutes, 75% ethanol for 5minutes, 50% ethanol for 5 minutes, and H2O for 5 minutes.
Antigen retrieval
Retrieve the epitopes by boiling the sections in Dako Antigen Retrieval Agent pH9 (Dako) for 20 minutes inside a boiling cuvette in a pressure cooker.
Allow slides to cool down within the retrieval solution for 1h at room temperature and then wash twice with PBS++.
Epitope dephosphorylation
Delimit the tissue sections with a fat pen (Dako pen).
Incubate tissue sections with 25U Antarctic Phosphatase in AP buffer, or with AP buffer only (vehicle control) for 2 hours at 37°C in a humidified chamber.
Blocking
Block unspecific binding sites with 5% BSA in PBS++ for 1h at room temperature in a humidified chamber. Ensure that the blocking solution covers the entire tissue section.
Proceed with standard immunostaining protocols.
Use freshly cut tissue sections, and evaluate their quality at a light microscope before staining.
Prepare all solutions freshly.
As a positive control, you may use a reliable phospho-epitope staining previously established in your laboratory.
Some phospho-epitopes may need longer phosphatase incubation time.
Total: 4 hours
Deparaffinization: 35 minutes
Antigen Retrieval: 20 minutes boiling plus 1 hour cooling
Epitope dephosphorylation: 2 hours
Blocking: 1 hour
In case of phospho-specific antibodies, phosphorylated epitopes should be detected in the vehicle treated samples (provided the target is indeed phosphorylated in the tissue analysed) but should not be detected anymore after adequate phosphatase treatment.
No conflict of interests to declare.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted 30 Jul, 2019
You are reading this latest protocol version