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Summary

Abstract

Introduction

Protocol

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Discussion

Acknowledgements

Materials

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Medicine

İskelet Kasında Lipid Damlacık İçeriğinin Lif Tipi ve Subhücresel-Spesifik Analizi

Published: June 8th, 2022

DOI:

10.3791/63718

1Endocrinology, Diabetes and Nutrition Unit, Institute of Experimental and Clinical Research, Medical Sector, Université Catholique de Louvain, 2Departamento de Fisiología, Facultad de Biología, Universidad de Sevilla

Artan kanıtlar, iskelet kası içindeki lipitlerin aşırı infiltrasyonunun lipotoksisite ve diyabet ile sonuçlandığını göstermektedir. Burada, lipit damlacıklarının boyutunu, yoğunluğunu ve hücre altı dağılımını lif tipine özgü bir şekilde ölçmek için doku işleme, Bodipy ile boyama, görüntü elde etme ve analiz dahil olmak üzere eksiksiz bir protokol sunuyoruz.

Miyosteatoz olarak bilinen iskelet kası lipid infiltrasyonu, obezite ve yaşlanma ile birlikte artar. Miyostoeatoz ayrıca son zamanlarda kardiyovasküler hastalık ve kanser gibi diğer birçok bozukluk için negatif bir prognostik faktör olarak keşfedilmiştir. Aşırı lipid infiltrasyonu kas kütlesini ve gücünü azaltır. Ayrıca total intramiyosellüler lipid içeriğine, lipid damlacık (LD) morfolojisine ve hücre altı dağılımına bağlı olarak lipotoksisite ve insülin direnci ile sonuçlanır. Lif tipi (oksidatif vs glikolitik) de önemlidir, çünkü oksidatif lifler lipitleri kullanmak için daha büyük bir kapasiteye sahiptir. Patofizyolojideki önemli etkileri nedeniyle, LD dinamiği ve fonksiyonu üzerine lif tipine özgü bir şekilde derinlemesine çalışmalar yapılması gerekmektedir.

Bu çalışmada, intramiyosellüler lipid içeriğinin ölçülmesi ve LD morfolojisinin ve hücre altı dağılımının lif tipine özgü bir şekilde analizi için eksiksiz bir protokol sunulmaktadır. Bu amaçla, seri kas kriyolitleri floresan boya Bodipy ve miyozin ağır zincir izoformlarına karşı antikorlar ile boyandı. Bu protokol, farklı kasların aynı anda işlenmesini, zamandan tasarruf edilmesini ve olası eserlerin önlenmesini sağlar ve Fiji'de oluşturulan kişiselleştirilmiş bir makro sayesinde LD analizinin otomatikleştirilmesi de mümkündür.

Miyosteatoz olarak bilinen iskelet kası lipid infiltrasyonu, obezite ve yaşlanma ile birlikte artar. Miyostoeatoz kas kütlesi ve gücü ile insülin duyarlılığı ile negatif ilişkilidir1. Ayrıca, son zamanlarda yapılan çalışmalar, miyosteatoz derecesinin kardiyovasküler hastalık2, alkolsüz yağlı karaciğer hastalığı3 veya kanser4 gibi diğer durumlar için prognostik bir faktör olarak kullanılabileceğini göstermektedir. Lipitler, kas lifleri arasındaki iskelet kasında ekstramiyosellüler lipitler olarak veya lifler içinde, hücre içi lipitler (IMCL'ler) olarak birikebilir. IMCL'ler ....

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Fareler üzerinde yürütülen tüm prosedürler, Université Catholique de Louvain'deki Tıp Sektöründen Hayvan Deneyleri Etik Komitesi tarafından onaylanmıştır (2019 / UCL / MD / 013).

1. Numunelerin dondurulması için diseksiyonu ve hazırlanması

  1. Her kas çifti için 3 mm kalınlığında bir mantar parçasını etiketleyin.
  2. Mantarın ortasına bir bıçakla yapılan küçük bir kesiden, dik olarak destek görevi görecek dikdörtgen bir sert pl.......

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Burada açıklanan protokol, LD'leri fiber tipinde ve hücre altına özgü bir şekilde kolayca ölçmek için etkili bir yöntem sağlar. EDL ve soleus gibi benzer büyüklükteki iki kasın birlikte dondurulmasıyla, aşağıdaki adımlara harcanan zaman ve kaynakların nasıl yarı yarıya azaldığını gösterir.

İmmün boyama, görüntü elde etme ve yetişkin fare kaslarında eksprese edilen farklı MyHC izoformlarının analizi için eksiksiz bir protokol sağlanır. Bu protokol, ilk.......

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Burada ayrıntılı olarak açıklanan protokol, Bodipy ile etiketlenmiş LD'leri fiber tipi ve hücre altı spesifik olarak ölçmek için etkili bir yöntemi açıklamaktadır. Son yıllarda, ORO veya Sudan Black B gibi klasik lipit boyaları, nötr lipitlere (örneğin, Bodipy) bağlanan yeni bir hücre geçirgen, lipofilik, floresan boya dizisi ile değiştirilmiştir. Farklı konjugatlar olarak mevcut olan Bodipy'nin, LD'leri morfolojilerini, dinamiklerini ve diğer organellerle etkileşimlerini incelemek için etik.......

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Bu çalışma, Fonds National de la Recherche Scientifique (FNRS-Crédit de Recherche J.0022.20) ve Société Francophone du Diabète (SFD-Roche Diabetes Care) tarafından verilen hibelerle desteklenmiştir. M.A.D.-L.d.C., Wallonie-Bruxelles Uluslararası Mükemmellik Programı'ndan burs aldı.

Yazarlar, Alice Monnier'e bu protokolün geliştirilmesine katkılarından dolayı ve Caroline Bouzin'e görüntü edinme sürecindeki uzmanlığı ve teknik yardımı için teşekkür eder. Ayrıca kriyostat ve mikroskoplara erişim için 2IP-IREC görüntüleme platformuna teşekkür ederiz (2IP-IREC Görüntüleme Platformu, Deneysel ve Klinik Araştırma Enstitüsü, Unive....

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NameCompanyCatalog NumberComments
Equipment
AxioCam 506 mono 6 Mpix cameraZeiss
AxioCam MRm 1.4MPix CCD camera Zeiss
Chemical hoodPotteau LaboEN-14175
Confocal microscopeZeissLSM800
Cork discs (ø 20 mm, 3 mm thick) Electron Microscopy Sciences63305
Cryo-GlovesTempshield16072252
Cryostat Thermo Scientific Microm Cryo Star HM 560
Dissecting Stereo Microscope SMZ745Nikon
Dry Ice
Dumont ForcepsF.S.T11295-10
Epifluorescence microscopeZeissAxioImage-Apotome Z1
Extra Fine Bonn ScissorsF.S.T14084-08
FisherBrand Disposable Base Molds (0.7 x 0.7 cm)ThermoFisher22-363-552Used to cut a piece to hold the muscle on the cork for freezing
Glass petri dish (H 25 mm, ø 150 mm)BRAND Petri dish, MERKBR455751Used to place the muscles on ice during dissection
ImmEdge Hydrophobic barrier PAP PenVector LabsH-4000Used to create an hidrophobic barrier around the muscle sections
IncubatorMMM MedcenterIncucell 707 
Microscope Cover Glasses (24x50 mm)Assistent 40990151
Microscope Slide Boxes Kartell278Used as humid chambers for immunohistochemistry
Neck holderLinie zwoSB-035X-02Used as strap to hold the stainless steel tumbler
No 15 Sterile Carbon Steel Scalpel BladeSwann-Morton0205
Paint brushesVan BleiswijckAmazon B07W7KJQ2XUsed to handle cryosections
Permanent Marker Pen BlackKlinipath/VWR98307-RUsed to label slides
Pierce Fixation ForcepsF.S.T18155-13
Polystyrene Box H 12 cm x L 25 cm x W 18 cm, used as a liquid nitrogen container and to transport the samples to the cryostat
Scalpel Handle, 125 mm (5"), No. 3AesculapBB073R
Stainless Steel Cup 10oz EboxerB07GFCBPFHTumbler to fill with isopentene for muscle freezing
Superfrost Ultra Plus slidesThermoFisherJ1800AMNZ
Surgical tweezers 1/2 teethMedische Vakhandel1303152Also called "Rat teeth tweezers"
Vannas Spring Scissors - 3 mm Cutting EdgeF.S.T15000-00
Weighing boatsVWR international611-2249
Whole-Slide Scanner for FluorescenceZeissAxio Scan.Z1
Reagents
Alexa Fluor 405 Goat Anti-Mouse IgG2bSigma-AldrichSAB4600477Used at a final concentration of 1:500
Alexa Fluor 488 Goat Anti-Mouse IgG1ThermoFisherA-21121Used at a final concentration of 1:500
Alexa Fluor 568 Goat Anti-Mouse IgMAbcamab175702Used at a final concentration of 1:1,000
Alexa Fluor 647 goat anti rat-IgG (H+L) secondary antibodyThermoFisherA-21247Used at a final concentration of 1:500
BODIPY-493/503 (4,4-difluoro-1,3,5,7,8-pentametil-4-bora-3a,4a-diaza-s-indaceno)ThermoFisherD3922Used at a final concentration of 1 µg/mL
BODIPY-558/568 C12 (4,4-Difluoro-5-(2-Thienyl)-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid)ThermoFisherD3835Used at a final concentration of 1 µg/mL
DAPI (4',6-diamidino-2-phenylindole)ThermoFisherD1306Used at a final concentration of 0.5 µg/mL
Dimethyl Sulfoxide (DMSO)Sigma-AldrichD-8418Used to solve Bodipy for the 1 mg/mL stock solution. CAUTION: Toxic and flammable. Vapors may cause irritation. Manipulate in a fume hood. Avoid direct contact with skin. Wear rubber gloves, protective eye goggles.
Formaldehyde solution 4%, buffered, pH 6.9Sigma-Aldrich1004969011CAUTION: May cause an allergic skin reaction. Suspected of causing genetic defects. May cause cancer. Manipulate in a fume hood. Avoid direct contact with skin. Wear rubber gloves, protective eye goggles.
Isopentane GPR RectaPurVWR international24872.298CAUTION: Extremely flammable liquid and vapor. May be fatal if swallowed and enters airways. May cause drowsiness or dizziness. Repeated exposure may cause skin dryness or cracking. Wear protective gloves/protective clothing/eye protection/face protection.
Liquid NitrogenCAUTION:  Extremely cold. Wear gloves. Handle slowly to minimize boiling and splashing and in well ventilated areas. Use containers designed for low-temperature liquids.
Mouse on mouse Blocking Reagent Vector LabsMKB-2213-1Used at concentration of 1:30
Myosin heavy chain Type I (BA-D5-s Primary Antibody) Gene: MYH7, monoclonal bovine anti mouse IgG2bDSHB University of IowaBA-D5-supernatantUsed at a final concentration of 1:10
Myosin heavy chain Type IIA (SC-71-s Primary Antibody) Gene:  MYH2, Monoclonal bovine anti mouse IgG1DSHB University of IowaSC-71-supernatantUsed at a final concentration of 1:10
Myosin heavy chain Type IIX (6H1-s Primary Antibody), Gene:  MYH1, Monoclonal rabbit anti mouse IgMDevelopmental Studies Hybridoma Bank, University of Iowa6H1-supernatantUsed at a final concentration of 1:5
Normal Goat Serum (NGS)Vector LabsS-1000
PBS 0.1 MCommonly used on histology laboratories
ProLong Gold Antifade MountantInvitrogen P36930
Rat anti-Laminin-2 (α-2 Chain) primary antibody (monoclonal)Sigma-AldrichL0663Used at a final concentration of 1:1,000
Tissue-Tek O.C.T compoundSakura 4583
Software
Adobe Illustrator CCAdobe Inc.Used to design the figures
Adobe PhotoshopAdobe Inc.Confocal software
BioRenderhttps://biorender.com/Used to design the figures
Fiji/ImageJhttps://imagej.net/software/fiji/Used to analyse the acquired images
Microsoft PowerPointMicrosoftUsed to reconstruct the histology of the whole muscle after scanning the fiber types
Zen Blue 2.6ZeissUsed to reconstruct the histology of the whole muscle after scanning the fiber types

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