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.     

An Optimized Histochemical Method to Assess Skeletal Muscle Glycogen and Lipid Stores Reveals Two Metabolically Distinct Populations of Type I Muscle Fibers

Skeletal muscle energy metabolism has been a research focus of physiologists for more than a century. Yet, how the use of intramuscular carbohydrate and lipid energy stores are coordinated during different types of exercise remains a subject of debate. Controversy arises from contradicting data from numerous studies, which used different methodological approaches. Here we review the “pros and cons” of previously used histochemical methods and describe an optimized method to ensure the preservation and specificity of detection of both intramyocellular carbohydrate and lipid stores. For optimal preservation of muscle energy stores, air drying cryosections or cycles of freezing-thawing need to be avoided. Furthermore, optimization of the imaging settings in order to specifically image intracellular lipid droplets stained with oil red O or Bodipy-493/503 is shown. When co-staining lipid droplets with associated proteins, Bodipy-493/503 should be the dye of choice, since oil red O creates precipitates on the lipid droplets blocking the light. In order to increase the specificity of glycogen stain, an antibody against glycogen is used. The resulting method reveals the existence of two metabolically distinct myosin heavy chain I expressing fibers: I-1 fibers have a smaller crossectional area, a higher density of lipid droplets, and a tendency to lower glycogen content compared to I-2 fibers. Type I-2 fibers have similar lipid content than IIA. Exhaustive exercise lead to glycogen depletion in type IIA and IIX fibers, a reduction in lipid droplets density in both type I-1 and I-2 fibers, and a decrease in the size of lipid droplets exclusively in type I-1 fibers.     

Adipophilin distribution and colocalisation with lipid droplets in skeletal muscle

Intramyocellular lipids (IMCL) are stored as discrete lipid droplets which are associated with a number of proteins. The lipid droplet-associated protein adipophilin (the human orthologue of adipose differentiation-related protein) is ubiquitously expressed and is one of the predominant lipid droplet-proteins in skeletal muscle. The aim of this study was to investigate the subcellular distribution of adipophilin in human muscle fibres and to measure the colocalisation of adipophilin with IMCL. Muscle biopsies from six lean male cyclists (BMI 23.4 ± 0.4, aged 31 ± 2 years, W _max 346 ± 8) were stained for myosin heavy chain type 1, IMCL, adipophilin and mitochondria using immunofluorescence and viewed with widefield and confocal fluorescence microscopy. The present study shows that like IMCL, the adipophilin content is ~twofold greater in type I skeletal muscle fibres and is situated in the areas between the mitochondrial network. Colocalisation analysis demonstrated that 61 ± 2% of IMCL contain adipophilin. Although the majority of adipophilin is contained within IMCL, 36 ± 4% of adipophilin is not associated with IMCL. In conclusion, this study indicates that the IMCL pool is heterogenous, as the majority but not all IMCL contain adipophilin.     

Correlation of individual skeletal muscle fibres from "semithin" sections stained with p-phenylene-diamine and histochemical sections incubated for myofibrillar ATP-ase.

Muscle fibres in transverse "semithin" sections of osmium-fixed and plastic embedded muscle tissue stained with p-phenylene-diamine has been classified into three types (M, INT and H) and correlated to the same fibres in neighboring sequential preincubated myofibrillar ATP-ase sections. A close correlation, but no equality was found between the fibre types classified according to the ATP-ase and the p-phenylene-diamine methods. On the average, the content of mitochondria and fat droplets was highest in the ATP-ase classified type I fibres (dominated by M fibres), and higher in the endurance trained than in the untrained subjects.     

Fine structure of single fibres of human skeletal muscle

Summary Individual muscle fibres were separated from freeze-dried needle biopsies and classed as type I or type II fibres according to their myofibrillar ATP-ase. Portions of the same fibres were processed for electron microscopy and their fine structure examined. Type I fibres were found to have thicker Z-bands and more mitochondria and lipid droplets than the type II fibres.     

Skeletal muscle characteristics of reindeer (Rangifer tarandus L.)

Fibre type composition, fibre areas, capillaries, enzyme activities and intramuscular substrates were analysed on skeletal muscle samples from reindeer. The muscles contained 10-20% Type I fibres and a higher percentage of Type IIB (40-60%) than Type IIA fibres (20-40%). All fibre types revealed medium or dark staining intensity for oxidative capacity. Glycolytic capacity was greatest in Type IIB fibres. All fibres stained for glycogen, while Type I and IIA fibres stained for lipids. The mean number of capillaries in contact with fibres of each type, relative to fibre type area was high in all muscle types. The metabolic profile of reindeer muscle indicates that energy, to a great extent, is produced through oxidative pathways.     

Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O.

Cultured 3T3-F442A cells differentiate into adipocytes and accumulate lipid droplets in the cytoplasm. When fat cells are stained with Oil red O, the degree of staining seems to be proportional to the extent of cell differentiation. We report here a fast and simple method to quantitate the extent of adipose conversion by staining the accumulated lipid with Oil red O and determining the amount of extracted dye at 510 nm. The results show that Oil red O specifically stains triglycerides and cholesteryl oleate but no other lipids. This technique is a valuable tool for processing large numbers of cell cultures or samples in which adipose differentiation and/or accumulated triglycerides is to be quantitated.     

An Orcein-Oil Red O Stain for Concomitant Demonstration of Elastic Tissue and Lipid

Orcein, 0.5% in 50% isopropanol, 0.5-1 hr, followed by saturated oil red O in isopropanol diluted 3:2 with distilled water, 10-15 min, was used to demonstrate lipids and elastic tissue simultaneously in 10 μ frozen sections of formalin-fixed aortas of the wild African buffalo, showing atherosclerotic lesions. A comparison was made with the oil red O-aldehyde fuchsin (AF) method of Kwaan and Hopkins (Stain Techn., 39: 123-5, 1964) and the resorcin fuchsin (RF)-oil red O method of Lillie (Histopathologic Technic and Practical Histochemistry, McGraw-Hill, 1954), but both gave marked background staining by AF or RF that obscured the smaller deposits of lipid. Sudan IV could be substituted for oil red but did not demonstrate many of the finest deposits of lipids. Sudan black, in combination with orcein, AF or RF, was very satisfactory for demonstrating lipids but obscured many elastic fibres. Sudan dyes I, II, III, brown, blue, and green, with orcein, AF or RF, showed less contrast between lipids and elastic tissue or failed to stain the lipids adequately.     

Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with oil red O

Summary Cultured 3T3-F442A cells differentiate into adipocytes and accumulate lipid droplets in the cytoplasm. When fat cells are stained with Oil red O, the degree of staining seems to be proportional to the extent of cell differentiation. We report here a fast and simple method to quantitate the extent of adipose conversion by staining the accumulated lipid with Oil red O and determining the amount of extracted dye at 510 nm. The results show that Oil red O specifically stains triglycerides and cholesteryl oleate but no other lipids. This technique is a valuable tool for processing large numbers of cell cultures or samples in which adipose differentiation and/or accumulated triglycerides is to be quantitated.     

Optimisation of oil red O staining permits combination with immunofluorescence and automated quantification of lipids

The objective of the present study was to develop a stain permitting automated quantification of myocellular lipid depositions in skeletal muscle sections together with immunolocalisation of other myocellular constituents by fluorescence microscopy. Lipid droplets were detected in skeletal muscle by oil red O (ORO). Conventional ORO was modified to diminish background staining, prevent crystallisation of ORO and to optimise lipid retention in cryosections. These modifications resulted in a punctate staining of lipid droplets, rather than the somewhat diffuse staining by conventional ORO. Small cavities in muscle sections (like the lumen of small blood vessels) lack ORO when using the protocol presented here. In addition a staining protocol is presented combining ORO with immunofluorescence. This combination permits multiple staining studies in the same section. Thus, lipid droplets can be studied together with immunolabelling of proteins involved in lipid handling and metabolism. This will extend our knowledge on the subcellular localisation of lipid handling proteins (i.e. enzymes and fatty acid transporting proteins) in relation to the localisation of lipid depositions. In conclusion, the protocol presented here permits examination of ORO-stained lipid droplets in skeletal muscle sections together with multiple staining of other immunodetectable proteins present in skeletal muscle by quantitative fluorescence microscopy.     

Correlation of individual skeletal muscle fibres from “semithin” sections stained with p-phenylene-diamine and histochemical sections incubated for myofibrillar ATP-ase

Summary Muscle fibres in transverse semithin sections of osmium-fixed and plastic embedded muscle tissue stained with p-phenylene-diamine has been classified into three types (M, INT and H) and correlated to the same fibres in neighboring sequential preincubated myofibrillar ATP-ase sections. A close correlation, but no equality was found between the fibre types classified according to the ATP-ase and the p-phenylene-diamine methods. On the average, the content of mitochondria and fat droplets was highest in the ATP-ase classified type I fibres (dominated by M fibres), and higher in the endurance trained than in the untrained subjects.     

A study of the peripheral innervation and muscle fibre types of Ictalurus nebulosus (Lesueur) and Ictalurus punctatus (Rafinesque)

The innervation pattern and fibre types of the axial musculature of two closely related catfish species with differing lifestyles, Ictalurus nebulosus (Lesueur) and I. punctatus (Rafinesque) were investigated. Both fish displayed the multiple innervation pattern in the red muscle. However, the white muscle of I. nebulosus demonstrated terminal innervation while I. puncrurus displayed multiply innervated white muscle fibres. Fibre typing utilizing histochemical techniques for glyco-gen, lipid, succinic dehydrogenase and glucose-6-phosphate dehydrogenase revealed the typical teleostean distribution of red, intermediate and white muscle fibres in both fish. Staining was greatest in the red muscle fibres and least in the white muscle fibres. The white muscle fibres of I. punctatus stained slightly more for lipid than the white fibres of I. nebulosus which may be correlated with a greater aerobic capacity related to lifestyle and possibly innervation.     

Fine structure and metabolic adaptation of red and white muscles in tuna

Synopsis An electron microscopic study of the red and white muscle fibres in the trunk musculature of the Kawakawa tuna (Euthynnus affinis) was carried out with a view to correlating their structure with metabolic adaptation. The red fibres which are considerably smaller in diameter (34.58 m ± 6.16 S.D.) are characterized by their high content of myoglobin, mitochondria, lipid droplets and glycogen granules. The white fibres which are relatively larger in diameter (66.03 m ± 11.59 S.D.) are characterized by their lack of myoglobin, low mitochondria) density, high content of glycogen granules and the conspicuous absence of lipid droplets. The characteristics in fine structure of the two fibre types are discussed in the light of their metabolic adaptation, the red fibres as being adapted for long term cruising movement utilizing lipid as the main source of energy and the white fibres for short bursts of activity metabolizing glycogen as the chief fuel.The tuna, with the acquisition of the counter-current heat exchange system which provides for the retention of the heat generated from high substrate oxidation in the red muscle and an efficient respiratory system, it is postulated, is well adapted for high speed sustained swimming.     

SFRP5抑制BMP9诱导人脐带间充质干细胞成骨分化的实验研究

目的:探讨脂肪因子分泌型卷曲相关蛋白(secreted frizzled-related protein 5,SFRP5)对骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)诱导人脐带间充质干细胞(human umbilical cord-derived mesenchymal stem cells,hUC-MSCs)成骨分化的影响。方法:将人脐带间充质干细胞根据不同的处理因素分为4组:对照组、BMP9组、BMP9+SFRP5组和SFRP5组;分别在3天、5天和7天进行碱性磷酸酶(alkaline phosphatase,ALP)活性读数,7天进行ALP染色,21天进行茜素红染色检测钙盐沉积及油红O染色;收集不同组的细胞用于裸鼠皮下注射成骨模型的建立,4周后取出离体骨进行Micro-CT扫描和分析,获取的标本进行HE、Masson染色,Alcian blue染色及油红O染色检测。Western blot检测成骨分化相关蛋白Runx2和OPN的表达。结果:BMP9组的ALP活性读数和染色结果和茜素红染色结果均较对照组增加,而BMP9+SFRP5组则较BMP9组降低;BMP9处理后出现少量脂滴,而BMP9+SFRP5组脂滴明显增加,SFRP5组脂滴最多;裸鼠皮下注射成骨模型的观察结果显示,对照组和SFRP5组没有形成肉眼可见的皮下包块,BMP9组和BMP9+SFRP5组能生成异位骨;4周后观测大体标本以及进行MicroCT检测,发现BMP9+SFRP5组的骨密度值小于BMP9组(P0.05)。HE、Masson染色,Alcian blue染色结果显示,BMP9组的骨分化程度大于BMP9+SFRP5,油红O染色结果示BMP9+SFRP5组有较多的成脂分化;SFRP5能抑制BMP9诱导的Runx2、OPN的蛋白质表达。结论:SFRP5抑制BMP9诱导的人脐带间充质干细胞成骨分化。     

Fibre types in the locomotory muscles of an antarctic teleost,Notothenia rossii

Summary The metabolic and structural differentiation of locomotory muscles of Notothenia rossii has been investigated. In this species sustained locomotion is achieved by sculling with enlarged pectoral fins (labriform locomotion), whilst the segmental myotomal muscle is reserved for burst activity. Red, white and subepidermal fibres can be distinguished in the trunk by histochemical and ultrastructural criteria. The main pectoral muscle (m. adductor profundus) consists entirely of red fibres. These three main fibres types show differences in histochemical staining profiles, capillarization, myofibril shape and packing, and lipid and mitochondrial content. The fractional volume of mitochondria amounts to 38% for pectoral, 30% for red myotomal and 1.9% for white myotomal fibres. Enzyme activities of red pectoral muscle are consistent with a higher potential for aerobic glucose and fatty acid oxidation than for the red myotomal fibres. Mg²⁺ Ca²⁺ -myofibrillar ATPase activities are similar for red pectoral and myotomal muscles and approximately half of those white fibres. Specialisations of N. rossii muscles associated with labriform swimming and locomotion at Antarctic temperatures are discussed.     

MiR-34a通过抑制Pgc-1β和线粒体生成促进小鼠骨骼肌脂肪异位沉积

过氧化物酶体增殖物激活受体γ辅激活因子-1β（peroxisome proliferative activated receptor γ coactivator 1 β,Pgc-1β）与线粒体生成相关。已有研究证明,miR-34a在肝组织脂肪异位沉积中发挥重要作用,但是否与骨骼肌的脂肪异位沉积相关尚不清楚。本研究以C57Bl/6J小鼠为研究对象,通过尾静脉注射miR-34a模拟物,探讨miR-34a过表达对小鼠骨骼肌脂肪沉积的影响。组织切片进行油红O染色及甘油三酯含量测定揭示,miR-34a过表达的小鼠骨骼肌组织中脂滴积累及甘油三酯含量显著增加。实时荧光定量PCR（qRT-PCR）显示,与对照鼠比较,miR-34a处理的小鼠骨骼肌组织中的脂肪酸合成酶(Fas）表达显著上调,而脂肪酸氧化分解相关基因产物肉毒碱棕榈酰基转移酶1α（Cpt 1α）表达显著下调,提示miR-34a调控骨骼肌内脂肪的沉积机制可能是通过促进脂肪酸生成和抑制脂肪酸分解实现的。qRT-PCR和Western印迹证明,miR-34a可抑制Pgc-1β蛋白的表达。CoxⅡ/28S比例（线粒体定量指标）测定提示,注射miR-34a模拟物导致小鼠骨骼肌线粒体数目显著下调。生物信息分析显示,Pgc-1β mRNA的3′-UTR存在 miR-34a的潜在识别位点,因此miR-34a可能通过靶向识别Pgc-1β的3′-UTR抑制Pgc-1β表达,从而抑制线粒体生成。上述结果证明,miR-34a能通过靶向抑制PGC-1β表达,抑制线粒体生成,继而减少脂肪酸氧化分解,导致骨骼肌脂肪沉积增加。此外,上调脂肪酸合成酶也可能是miR-34a导致骨骼肌脂肪沉积增加的另一原因,其作用机制需进一步研究。     

The fine structure of pigeon breast muscle

The pectoralis major muscle of the pigeon is composed of two types of muscle fibre. In the Type I fibres, the myofibrils are closely packed and there are few mitochondria. The myofibrils in the Type II fibres are separated by numerous columns of large mitochondria and lipid droplets. The membrane systems of the two types of fibre are similar. The triads occur at the Z-line; the sarcoplasmic reticulum is in the form of large terminal cisternae which are joined by narrow longitudinal tubules to a broad central cisterna. The value of morphological criteria in the classification of muscle fibres is discussed.     

Deformation of lipid droplets in fixed samples

Nile red, Sudan III, and oil red O have been used to stain lipid droplets (LDs) for fluorescence microscopy. We noticed that LDs labeled by Nile red are different in appearance from those stained by the latter two dyes. To understand the cause of the difference, we used sequential labeling procedures (first LD stain-photography-quenching-second LD stain-photography), and examined the effect of several factors. Immunofluorescence labeling for adipose differentiation-related protein (ADRP), an LD marker, was also observed comparatively with the lipid stains. As a result, we found that ethanol and isopropanol used for Sudan III and oil red O staining, respectively, and glycerol used for mounting, cause fusion of adjacent LDs even in glutaraldehyde-fixed samples. By the same treatment, immunofluorescence labeling for ADRP was dislocated to the rim of large LDs that were formed as a result of the artifactual fusion. The result indicates that the LD structure can be better observed with Nile red than with Sudan III or oil red O.     

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.     

Variations in fibre composition of the gastrocnemius muscle in rats subjected to speed training

Thirty-six adult Wistar rats were divided into three groups. One group was used as a control, and the other two underwent different training programmes in which greater relevance was attached to the intensity of exercise than to its duration. Samples of the red and mixed portions of m. gastrocnemius (caput lateralis) were stained with m-ATPase to determine the percentage of type I, IIA and IIB fibres, and with NADH-TR in order to quantify variations in the percentage of low staining intensity (FG) fibres. The most notable results obtained were: a) the ratio of type I type II fibres remained unchanged; b) the proportion of IIA fibres increased, while that of IIB fibres decreased correspondingly; c) FG fibres, which were virtually absent from the red portion, recorded a clear decrease which was more marked, and occurred more rapidly, than in IIB fibres. These differences were all statistically significant in the mixed portion of the muscle. Adaptive changes in fibre composition in the red portion were less marked.