Protocol

Part of the series Methods in Molecular Biology pp 1-14

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Immunohistochemical Detection of Sphingosine-1-Phosphate and Sphingosine Kinase-1 in Human Tissue Samples and Cell Lines

  • Gary M. ReynoldsAffiliated withCentre for Liver Research and NIHR Biomedical Research Unit, University of Birmingham and Queen Elizabeth Hospital Birmingham Email author 
  • , Barbara VisentinAffiliated withLa Jolla Biologics Inc
  • , Roger SabbadiniAffiliated withStanford University School of Medicine

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Abstract

Sphingosine-1-phosphate (S1P) and the enzyme primarily responsible for its production, sphingosine kinase-1 (SphK-1), are dysregulated in multiple human diseases including cancer, multiple sclerosis (MS), diabetes, neurological diseases, fibrosis, and certain pathologies associated with impaired angiogenesis such as age-related macular degeneration (AMD). Antibody-based techniques to identify and localize S1P and SphK-1 within cells and tissue specimens represent a powerful tool, not only to understand biological role of these molecules but also to validate these unique in-class targets in multiple state diseases. Consequently, the potential applications of these molecules for therapy and diagnostic purposes are currently under investigation. Here, we describe a new improved technique, Agitated Low Temperature Epitope Retrieval (ALTER) for staining procedures, to identify expression of S1P and SphK-1 in human frozen tissue samples. The challenges encountered in the process of localization in tissue samples of lipid molecules such as S1P are discussed.

Keywords:

Bioactive lipids S1P SPHK1 Anti-S1P antibody LT002 ALTER technique Immunohistochemistry Liver cancer