Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate

Poly-beta-hydroxybutyrate granules exhibited a strong orange fluorescence when stained with Nile blue A. Heat-fixed cells were treated with 1% Nile blue A for 10 min and were observed at an excitation wavelength of 460 nm. Glycogen and polyphosphate did not stain. Nile blue A appears to be a more specific stain for poly-beta-hydroxybutyrate than Sudan black B.     

Nile blue A as a fluorescent stain for poly-beta-hydroxybutyrate.

Poly-beta-hydroxybutyrate granules exhibited a strong orange fluorescence when stained with Nile blue A. Heat-fixed cells were treated with 1% Nile blue A for 10 min and were observed at an excitation wavelength of 460 nm. Glycogen and polyphosphate did not stain. Nile blue A appears to be a more specific stain for poly-beta-hydroxybutyrate than Sudan black B.     

Apolipoprotein A-V association with intracellular lipid droplets

Apolipoprotein A-V (apoA-V) plays a key role in the regulation of triglyceride (TG) metabolism. Given the very low concentration of apoA-V in plasma, we hypothesized that apoA-V may influence plasma TG levels by affecting the assembly and/or secretion of apoB-containing lipoproteins. When apoA-V was overexpressed in cultured Hep3B cells, neither the amount of apoB secreted nor the density distribution of apoB-containing lipoproteins was affected. Fluorescence microscopy and cell lysate immunoprecipitation studies revealed that apoA-V is not associated with apoB intracellularly, yet immunoprecipitation of apoA-V from the cell culture medium resulted in coprecipitation of apoB. These data suggest that the apoA-V association with apoB-containing lipoproteins is a postsecretory event. Confocal fluorescence microscopy revealed the presence of apoA-V in distinct cellular structures. Based on Nile Red staining, we identified these structures to be intracellular lipid droplets. These data suggest that apoA-V has a unique association with cellular lipids and, therefore, may be involved in the storage or mobilization of intracellular lipids.     

New technique to quantify the lipid composition of lipid droplets in porcine oocytes and pre-implantation embryos using Nile Red fluorescent probe

The principal objective of this study was to develop a novel method based on confocal microscopy and a solvatochromic fluorescent dye, Nile red (NR) to quantify the main types of lipids in a single mammalian oocyte and embryo. We hypothesize that NR staining followed by the decomposition of NR-spectra identifies and quantifies the triglycerides, phospholipids, and cholesterol in a single oocyte and embryo. We analyzed the lipid droplets in porcine oocytes and pre-implantation embryos up to the hatched blastocyst stage developed in vivo and in cultured blastocysts. The emission spectrum of NR-stained mixture of different lipid types is a convolution of several component spectra. The principal component analysis (PCA) and a multivariate curve resolution-alternating least squares method (MCR-ALS) allowed to decompose the emission spectrum and quantify the relative amount of each lipid type present in mixture. We reported here that the level of the triglycerides, phospholipids and cholesterol in lipid droplets significantly decreases by 17.7%, 26.4% and 23.9%, respectively, from immature to mature porcine oocytes. The content of triglycerides and phospholipids remains unchanged in droplets of embryos from the zygote up to the morula stage. Then the triglyceride level decreases in the blastocyst by 15.1% and in the hatched blastocyst by 37.3%, whereas the amount of phospholipids decreases by 10.5% and 12.5% at the blastocyst and hatched blastocyst stages, respectively. In contrast, the content of cholesterol in droplets does not change during embryo cleavage. The lipid droplets in the blastocyst produced in vivo contain lower amounts of triglycerides (by 26.1%), phospholipids (by 14.2%) and cholesterol (by 34.8%) than those in the blastocyst cultured in NCSU-23 medium. In conclusion, our new technique is suitable to quantify the content of triglycerides, phospholipids and cholesterol in individual mammalian oocytes and embryos. Our findings indicate an important role for lipids during porcine oocyte maturation and early embryonic development, and suggest an altered lipid metabolism in cultured embryos.     

Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O

Nile red is a phenoxazone dye that fluoresces intensely, and in varying color, in organic solvents and hydrophobic lipids. However, the fluorescence is fully quenched in water. The dye acts, therefore, as a fluorescent hydrophobic probe. We utilized this novel property of nile red to develop a sensitive fluorescent histochemical stain for tissue lipids. Nile red was prepared by boiling Nile blue A under reflux for 2 hr in 0.5% H2SO4, and extracting the product into xylene. For staining, the purified dye is dissolved in 75% glycerol (1-5 micrograms/ml) and applied to frozen tissue sections. Tissue lipids then fluoresce yellow-gold to red, depending on their relative hydrophobicity. Using sections of liver and aorta from a cholesterol-fed rabbit, we assessed the value of Nile red as a stain for neutral lipids by comparing the staining pattern obtained with that produced by oil red O, a commonly used dye for tissue cholesteryl esters and triacylglycerols. In the cholesterol fatty liver, Nile red staining was comparable to that of oil red O. In contrast, Nile red staining of rabbit aortic atheroma revealed ubiquitous lipid deposits not observed with oil red O staining. These latter results suggest that Nile red can detect neutral lipid deposits, presumably unesterified cholesterol, not usually seen with oil red O or other traditional fat stains.     

Nile red as a polarity-sensitive fluorescent probe of hydrophobic protein surfaces

Nile red is an uncharged hydrophobic molecule whose fluorescence is strongly influenced by the polarity of its environment. It interacts with many, but not all, native proteins, including beta-lactoglobulin, kappa-casein, and albumin, with a wide range of spectral changes for different proteins. It detects the exposure or formation of new hydrophobic surfaces induced by ligand binding to calmodulin, oligomerization of melittin, or unfolding of ovalbumin during early thermal denaturation. The dye is photostable, the working wavelength range is broad and removed from those at which many biomolecules absorb, the fluorescence is unaffected by pH between 4.5 and 8.5, the quantum yield is high, and hydrophobic sites on proteins may be investigated in dilute solutions.     

The use of a fluorescent dye, Nile red, to evaluate the lipid content of single mammalian oocytes

This study aimed to investigate the use of Nile red, a fluorescent dye specific for intracellular lipid droplets, to quantify the lipid content of single mammalian oocytes. It was hypothesized that a higher amount of lipid present in lipid droplets in an oocyte would result in a higher amount of emitted fluorescent light. Following fixation and subsequent staining of denuded oocytes, the fluorescence of the whole oocyte was visualized by fluorescence microscopy and quantified with a photometer and photomultiplier connected to the microscope. The peak of fluorescence was observed in the yellow spectrum (590 nm) and the fluorescence was restricted to the lipid droplets corresponding to apolar lipids. Nile red concentrations ranging from 0.1 to 10 microg/ml yielded similar results. After fixation, a minimum of 2 h staining was necessary to reach maximal fluorescence which remained stable for several hours. The position of the microscopic focus within the oocyte had no influence on the amount of measured fluorescence. Successive measurements of the same oocyte yielded very similar results indicating the repeatability of the method. Finally, the technique was validated by comparing the lipid content of bovine, porcine and murine immature oocytes, which are known to contain different amounts of lipids. After staining, the fluorescence of murine oocytes was 2.8-fold lower than the fluorescence of bovine oocytes which in turn were 2.4 times less fluorescent than porcine oocytes. Based on this study, it can be said that this rather fast and easy technique allows for the relative quantification of the lipid content (present in the lipid droplets) of one single oocyte. The different amounts of emitted fluorescent light in bovine, porcine and murine oocytes correlated with the known lipid contents in these three species. This technique could be used to compare the lipid content of oocytes originating from different donors, from different sized follicles or cultured in various conditions.     

Determining intracellular lipid content of different oleaginous yeasts by one simple and accurate Nile Red fluorescent method

Abstract

A simple and accurate Nile Red fluorescent method was built to evaluate the lipid content of three different oleaginous yeasts by one standard curve. The staining of cells can be observed clearly by laser scanning confocal microscope, showing that Nile Red can enter into the cells of oleaginous yeasts easily. A series of conditions such as pretreating temperature, cell suspension concentration (OD₆₀₀), staining time, Nile Red concentration and the type of suspension solvent were learnt systematically to obtain the optimal process parameters for Nile Red staining. After optimization, the fitting curve of Nile Red fluorescent method was established under suitable conditions (pretreating temperature: 50?°C, OD₆₀₀: 1.0; staining time: 5?mins; Nile Red concentration: 1.0?μg/mL; suspension solvent: PBS) and it had a suitable correlation coefficient (R² = 0.95) for lipid content measurement of different oleaginous yeasts. By this study, the possibility of lipid content determination of different oleaginous yeasts by one fitting curve can be proven and this will improve the efficiency of researches related to microbial lipid production.     

X-34, a fluorescent derivative of Congo red: a novel histochemical stain for Alzheimer's disease pathology.

X-34, a lipophilic, highly fluorescent derivative of Congo red, was examined as a histochemical stain for pathological changes in Alzheimer's disease (AD). X-34 intensely stained neuritic and diffuse plaques, neurofibrillary tangles (NFTs), neuropil threads, and cerebrovascular amyloid. Comparison to standard methods of demonstrating AD pathology showed that X-34 correlated well with Bielschowsky and thioflavin-S staining. X-34 staining of NFTs correlated closely with anti-TAU antibody staining. A 1:1 correspondence of X-34 and anti-A beta antibody staining of plaques and cerebrovascular amyloid was observed. Both X-34 and thioflavin-S staining were eliminated by formic acid pretreatment, suggesting that beta-sheet secondary protein structure is a necessary determinant of staining. X-34 may be a general amyloid stain, like Congo red, because it also stains systemic amyloid deposits due to lambda-light chain monoclonal gammopathy. In conclusion, X-34 is a highly fluorescent marker for beta-sheet structures and intensely labels amyloid plaques, NFTs, neuropil threads, and vascular amyloid in AD brains. It can be used with both paraffin-embedded and frozen tissues as well as in combination with immunohistochemistry for double labeling. The intensity of staining and the simplicity and reproducibility of the technique suggest that it may be a useful addition to the standard techniques for evaluation of AD neuropathology. (J Histochem Cytochem 48:1223-1232, 2000)     

The dynamic roles of intracellular lipid droplets: from archaea to mammals

During the past decade, there has been a paradigm shift in our understanding of the roles of intracellular lipid droplets (LDs). New genetic, biochemical and imaging technologies have underpinned these advances, which are revealing much new information about these dynamic organelles. This review takes a comparative approach by examining recent work on LDs across the whole range of biological organisms from archaea and bacteria, through yeast and Drosophila to mammals, including humans. LDs probably evolved originally in microorganisms as temporary stores of excess dietary lipid that was surplus to the immediate requirements of membrane formation/turnover. LDs then acquired roles as long-term carbon stores that enabled organisms to survive episodic lack of nutrients. In multicellular organisms, LDs went on to acquire numerous additional roles including cell- and organism-level lipid homeostasis, protein sequestration, membrane trafficking and signalling. Many pathogens of plants and animals subvert their host LD metabolism as part of their infection process. Finally, malfunctions in LDs and associated proteins are implicated in several degenerative diseases of modern humans, among the most serious of which is the increasingly prevalent constellation of pathologies, such as obesity and insulin resistance, which is associated with metabolic syndrome.     

The obligate intracellular pathogen Chlamydia trachomatis targets host lipid droplets

Lipid droplets (LDs) are ubiquitous but poorly understood neutral-lipid-rich eukaryotic organelles that may participate in functions as diverse as lipid homeostasis, membrane traffic, and signaling . We report that infection with the obligate intracellular pathogen Chlamydia trachomatis, the causative agent of trachoma and many sexually transmitted diseases , leads to the accumulation of neutral-lipid-rich structures with features of LDs at the cytoplasmic surface of the bacteria-containing vacuole. To identify bacterial factors that target these organelles, we screened a collection of yeast strains expressing GFP-tagged chlamydial ORFs and identified several proteins with tropism for eukaryotic LDs. We determined that three of these LD-associated (Lda) proteins are translocated into the mammalian host and associate with neutral-lipid-rich structures. Furthermore, the stability of one Lda protein is dependent on binding to LDs, and pharmacological inhibition of LD formation negatively impacted chlamydial replication. These results suggest that C. trachomatis targets LDs to enhance its survival and replication in infected cells. The co-option of mammalian LD function by a pathogenic bacterium represents a novel mechanism of eukaryotic organelle subversion and provides unique research opportunities to explore the function of these understudied organelles.     

Application of nile blue and nile red, two fluorescent probes, for detection of lipid droplets in human skeletal muscle

Using frozen sections from human muscle biopsies, we assessed the value of Nile blue and Nile red, two fluorescent probes, as stains for lipid droplets in normal and pathological skeletal muscle fibers. In normal muscle, lipid storage disorders, and mitochondrial myopathies, Nile blue stained the lipid droplets as yellow-gold fluorescent structures. The lipid droplets were also seen as yellow-gold fluorescent structures in Nile red-stained sections, but the outstanding feature in these preparations was the staining of the membrane network of the muscle fibers and membrane proliferations in pathological muscle as red-orange fluorescent structures. These results suggest that both Nile blue and Nile red stains are useful for visualization of lipid droplets and membrane proliferations in pathological muscle biopsies.     

Pyronin Y as a fluorescent stain for paraffin sections

Pyronin Y has long been used, in combination with other dyes such as Methyl Green, as a differential stain for nucleic acids in paraffin tissue sections. It also forms fluorescent complexes with double-stranded nucleic acids, especially RNA, enabling semi-quantitative analysis of cellular RNA in flow cytometry. However, the possibility of using pyronin Y as a fluorescent stain for paraffin tissue sections has rarely been investigated. We herein report that in sections stained with Methyl Green–pyronin Y, red blood cells, elastic fibre of blood vessels, zymogen granules of pancreatic acinar cells, surface membrane of heptocytes and kidney tubular cells showed strikingly strong green and/or red fluorescence, while the nuclei of cells appeared non-fluorescent. The use of confocal laser-scanning microscope greatly improved the resolution and selectivity of the fluorescent images. Staining with pyronin Y alone gave similar results in terms of fluorescence properties of the specimens. Pretreatment of paraffin sections with RNase significantly reduced cytoplasmic pyronin Y staining as judged by transmission light microscopy, but it had little effect on the fluorescence intensity of red blood cells, elastic fibres and zymogenbreak granules.     

Imaging of lipid biosynthesis: how a neutral lipid enters lipid droplets

The biosynthesis and storage of triglyceride (TG) is an important cellular process conserved from yeast to man. Most mammalian cells accumulate TG in lipid droplets, most prominent in adipocytes, which are specialized to store large amounts of the TG over long periods. In this study, we followed TG biosynthesis and targeting by fluorescence imaging in living 3T3-L1 adipocytes and COS7 fibroblasts. Key findings were (i) not only TG but also its direct metabolic precursor diacylglycerol, DG, accumulates on lipid droplets; (ii) the essential enzyme diacylglycerol acyltransferase 2 associates specifically with lipid droplets where it catalyzes the conversion of DG to TG and (iii) individual lipid droplets within one cell acquire TG at very different rates, suggesting unequal access to the biosynthetic machinery. We conclude that at least part of TG biosynthesis takes place in the immediate vicinity of lipid droplets. In vitro assays on purified lipid droplets show that this fraction of the biosynthetic TG is directly inserted into the growing droplet.     

A high throughput Nile red fluorescence method for rapid quantification of intracellular bacterial polyhydroxyalkanoates

A rapid quantitative measurement of accumulated polyhydroxyalkanoate (PHA) is essential for rapid monitoring of PHA production by microorganisms. In the present study, a 96-well microplate was used as a high throughput means to measure the fluorescence intensity of the Nile red stained cells containing PHA. The linear correlation obtained between intracellular PHA concentration and the fluorescence intensity represents the potential of the Nile red method employment to determine PHA concentration. The optimal ranges of excitation and emission wavelengths were determined using bacterial cells containing different types of PHAs, of different co-monomers and compositions. Interestingly, in spite of different co-monomers compositions in each PHA, all tested PHAs fluoresced maximally at excitation wavelength between 520 and 550 nm, and emission wavelength between 590 and 630 nm. The developed staining method also had successfully demonstrated a good correlation between the amount of accumulated PHA based on the fluorescence intensity measurements and that from chromatographic analysis to evaluate poly(3-hydroxybutyrate) [P(3HB)], poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)], using the same calibration curve, despite of different co-monomers that the PHA consist. Strongly supported by these experimental results, it can therefore be concluded that the developed staining method can be efficiently applied for rapid monitoring of PHA production.