This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Dephosphorylation of epitopes in murine skin sections
Sandra Iden
CECAD, University of Cologne
Corresponding Author
ORCiD: https://orcid.org/0000-0003-2333-9827
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
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.
Keywords
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.
Version History
CURRENT STATUS: Posted
Version 1
Posted 30 Jul, 2019
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Associated Publications
Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity
by Martim Dias Gomes, Soriba Letzian, Michael Saynisch, +1
Nature Communications (29 July, 2019)
Method Article
Dephosphorylation of epitopes in murine skin sections
Sandra Iden
CECAD, University of Cologne
Corresponding Author
ORCiD: https://orcid.org/0000-0003-2333-9827
Version History
CURRENT STATUS: Posted
Version 1
Posted 30 Jul, 2019
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
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.
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.
Associated Publications
Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity
by Martim Dias Gomes, Soriba Letzian, Michael Saynisch, +1
Nature Communications (29 July, 2019)