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An optimized method for Oil Red O staining with the salicylic acid ethanol solution.

ABSTRACT: Oil Red O (ORO) staining is a commonly used experimental technique to detect lipid content in cells or tissues. Freshly prepared ORO in 60% isopropanol is the most widely used method at present. However, isopropanol is volatile and harmful to the human body. It will also affect the interpretation of the results due to the formation of crystals and non-specific diffuse staining. In this paper, by screening and validation, we report a salicylic acid ethanol solution (containing 50% ethanol, 5%-10% salicylic acid) for the preparation of ORO solution, which has a better staining effect on lipid staining in cells and tissues, with a clean background and short dyeing time. What's more, this ORO solution is non-toxic, convenient to prepare, and can be stored for a long time. Therefore, it is reliable, easy to operate, and can be widely popularized and applied in laboratories.

SUBMITTER: Du J

PROVIDER: S-EPMC9980477 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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An optimized method for Oil Red O staining with the salicylic acid ethanol solution.

Du Junbao J Zhao Li L Kang Quan Q He Yun Y Bi Yang Y

Adipocyte 20231201 1

Oil Red O (ORO) staining is a commonly used experimental technique to detect lipid content in cells or tissues. Freshly prepared ORO in 60% isopropanol is the most widely used method at present. However, isopropanol is volatile and harmful to the human body. It will also affect the interpretation of the results due to the formation of crystals and non-specific diffuse staining. In this paper, by screening and validation, we report a salicylic acid ethanol solution (containing 50% ethanol, 5%-10% ...[more]

PMID: 36779587

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Project description:BackgroundSalicylic acid (SA) is a key defense signal molecule against biotrophic pathogens in plants. Quantification of SA levels in plants is critical for dissecting the SA-mediated immune response. Although HPLC and GC/MS are routinely used to determine SA concentrations, they are expensive and time-consuming. We recently described a rapid method for a bacterial biosensor Acinetobacter sp. ADPWH_lux-based SA quantification, which enables high-throughput analysis. In this study we describe an improved method for fast sample preparation, and present a high-throughput strategy for isolation of SA metabolic mutants.ResultsOn the basis of the previously described biosensor-based method, we simplified the tissue collection and the SA extraction procedure. Leaf discs were collected and boiled in Luria-Bertani (LB), and then the released SA was measured with the biosensor. The time-consuming steps of weighing samples, grinding tissues and centrifugation were avoided. The direct boiling protocol detected similar differences in SA levels among pathogen-infected wild-type, npr1 (nonexpressor of pathogenesis-related genes), and sid2 (SA induction-deficient) plants as did the previously described biosensor-based method and an HPLC-based approach, demonstrating the efficacy of the protocol presented here. We adapted this protocol to a high-throughput format and identified six npr1 suppressors that accumulated lower levels of SA than npr1 upon pathogen infection. Two of the suppressors were found to be allelic to the previously identified eds5 mutant. The other four are more susceptible than npr1 to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 and their identity merits further investigation.ConclusionsThe rapid SA extraction method by direct boiling of leaf discs further reduced the cost and time required for the biosensor Acinetobacter sp. ADPWH_lux-based SA estimation, and allowed the screening for npr1 suppressors that accumulated less SA than npr1 after pathogen infection in a high-throughput manner. The highly efficacious SA estimation protocol can be applied in genetic screen for SA metabolic mutants and characterization of enzymes involved in SA metabolism. The mutants isolated in this study may help identify new components in the SA-related signaling pathways.

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An optimized method for Oil Red O staining with the salicylic acid ethanol solution.

Publications

An optimized method for Oil Red O staining with the salicylic acid ethanol solution.

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