Format

Send to

Choose Destination
J Vis Exp. 2018 Jun 21;(136). doi: 10.3791/57760.

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue.

Author information

1
Department of Molecular, Cell and Developmental Biology, UCLA; Department of Biological Chemistry, David Geffen School of Medicine.
2
Department of Molecular, Cell and Developmental Biology, UCLA; Molecular Biology Institute Interdepartmental Program, UCLA.
3
Department of Molecular, Cell and Developmental Biology, UCLA.
4
Department of Biological Chemistry, David Geffen School of Medicine.
5
Department of Biological Chemistry, David Geffen School of Medicine; Molecular Biology Institute Interdepartmental Program, UCLA.
6
Department of Molecular, Cell and Developmental Biology, UCLA; Department of Biological Chemistry, David Geffen School of Medicine; Molecular Biology Institute Interdepartmental Program, UCLA; hcoller@ucla.edu.

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.

PMID:
29985359
PMCID:
PMC6101961
DOI:
10.3791/57760
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for MyJove Corporation Icon for PubMed Central
Loading ...
Support Center