Mapping metabolism: Monitoring lactate dehydrogenase activity directly in tissue

David Jelinek, Aimee Flores, Melanie Uebelhoer, Vincent Pasque, Kathrin Plath, M. Luisa Iruela-Arispe, Heather R. Christofk, William E. Lowry, Hilary A. Coller*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

Original languageEnglish (US)
Article numbere57760
JournalJournal of Visualized Experiments
Volume2018
Issue number136
DOIs
StatePublished - Jun 21 2018

Keywords

  • Biochemistry
  • Enzymatic activity
  • In situ
  • Issue 136
  • Lactate dehydrogenase
  • Metabolic map
  • Metabolism
  • Mouse skin

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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