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Hypertrophy Analysis and Quantification in Embryonic Cardiomyocites
胚胎心肌细胞的肥大分析和量化   

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Abstract

Myocardial growth goes from proliferation to hypertrophy during development. The measurement of the relative cell area provides information of cardiomyocyte hypertrophy, which is ideal for studying myocardial development.

Material and Reagents

  1. Mouse embryos
  2. 4-6 μm paraffin sections of embryonic hearts
  3. 4% PFA
  4. Paraffin
  5. Xylene (Merck KGaA, catalog number: 8.08691.1000 )
  6. Ethanol (Merck KGaA, catalog number: 1.00983.1000 )
  7. PBS
  8. Wheat germ agglutinin (WGA) conjugates tetramethylrhodamine (Life Technologies, Molecular Probes®, catalog number: W849 ) (1:100)
  9. DAPI (Invitrogen, catalog number: D1306 )
  10. Distilled water
  11. Fluorescence mounting media (Fluoromont-G) (SouthernBiotech, catalog number: 0100-01 )

Equipment

  1. Microtome
  2. Fluorescence microscope
  3. Humid chamber

Software

  1. Image analysis software (Image J)

Procedure

  1. Embryos are fixed in 4% PFA and embedded in paraffin. (Mouse embryos are fixed ON at 4 °C and embedded in paraffin after dehydration with Ethanol and Xylene. E16.5 embryos were dehydrated 45 min ethanol 50%, 45 min ethanol 70%, 45 min ethanol 80%, 45 min ethanol 90%, 45 min ethanol 95%, 30 min ethanol 100%, 30 min ethanol 100%, 30 min xylene and 3 times 1-hour paraffin before orientation. Everything at room temperature but the paraffin which is done in a stove at 65 °C)
  2. Rehydrate the sections (65 °C 20 min, xylene 5 min, xylene 5 min, ethanol 100% 5 min, ethanol 100% 5 min, ethanol 96% 5 min, ethanol 90% 5 min, water 5 min).
  3. Wash with Distilled water 5 minutes three times at room temperature.
  4. Wash with 1x PBS 5 min twice at room temperature.
  5. Incubate WGA (1:100) 45 min at room temperature in humid chamber.
  6. Wash with 1x PBS 5 min three times.
  7. Incubate with DAPI (1:1,000) 10 min at room temperature in humid chamber and in darkness.
  8. Wash with 1x PBS 5 min twice.
  9. Mount with fluorescence mounting media (store at 4 °C in darkness before analysis).
  10. Make pictures under the microscope.
  11. Analyse pictures measuring the cell area using ImageJ. Measure at least 100 cells per section and three sections per embryo. Below image example of compact myocardium with the membranes stained with WGA where the relative area was measured. Myocites are identified ignoring the endocardial and endothelial cells, these cells form a monolayer covering the cardiomyocites. Cells within the myocardial wall are measured.


    Figure 1. 7 µm transverse sections of a E15.5 WT heart stained with fluorophore-coupled wheat-germ agglutinin (WGA)

Acknowledgments

This study was funded by grants SAF2010-17555, RD06/0014/0038 (RECAVA) and, RD06/0010/1013 (TERCEL) from the Spanish Ministry of Economy and Competition (MINECO) and EU FP7-ITN 215761 (NotchIT) to J.L.d.l.P. G.L. had a PhD fellowship from the MINECO (FPI Program, BES-2008-002904).

References

  1. Luxan, G., Casanova, J. C., Martinez-Poveda, B., Prados, B., D'Amato, G., MacGrogan, D., Gonzalez-Rajal, A., Dobarro, D., Torroja, C., Martinez, F., Izquierdo-Garcia, J. L., Fernandez-Friera, L., Sabater-Molina, M., Kong, Y. Y., Pizarro, G., Ibanez, B., Medrano, C., Garcia-Pavia, P., Gimeno, J. R., Monserrat, L., Jimenez-Borreguero, L. J. and de la Pompa, J. L. (2013). Mutations in the NOTCH pathway regulator MIB1 cause left ventricular noncompaction cardiomyopathy. Nat Med 19(2): 193-201.

简介

心肌生长在发育期间从增殖到肥大。 相对细胞面积的测量提供心肌细胞肥大的信息,这是研究心肌发育的理想选择。

材料和试剂

  1. 小鼠胚胎
  2. 胚胎心脏的4-6μm石蜡切片
  3. 4%PFA
  4. 石蜡
  5. 二甲苯(Merck KGaA,目录号:8.08691.1000)
  6. 乙醇(Merck KGaA,目录号:1.00983.1000)
  7. PBS
  8. 小麦胚凝集素(WGA)缀合物四甲基罗丹明(Life Technologies,Molecular Probes ,目录号:W849)(1:100)
  9. DAPI(Invitrogen,目录号:D1306)
  10. 蒸馏水
  11. 荧光封固介质(Fluoromont-G)(SouthernBiotech,目录号:0100-01)

设备

  1. 切片机
  2. 荧光显微镜
  3. 湿度室

软件

  1. 图像分析软件(Image J)

程序

  1. 将胚胎固定在4%PFA中并包埋在石蜡中。 E16.5胚胎脱水45分钟乙醇50%,45分钟乙醇70%,45分钟乙醇80%,45分钟乙醇90%脱水45分钟乙醇和二甲苯脱水后,将石蜡包埋在石蜡中(小鼠胚胎在4℃固定ON) ,45分钟乙醇95%,30分钟乙醇100%,30分钟乙醇100%,30分钟二甲苯和3次取向前1小时的石蜡,一切于室温,但石蜡在65℃的炉中进行)
  2. 再水合切片(65℃20分钟,二甲苯5分钟,二甲苯5分钟,乙醇100%5分钟,乙醇100%5分钟,乙醇96%5分钟,乙醇90%5分钟,水5分钟)。
  3. 在室温下用蒸馏水洗涤5分钟三次。
  4. 用1x PBS在室温下洗涤两次,每次5分钟
  5. 在室温下在湿室中孵育WGA(1:100)45分钟
  6. 用1x PBS洗涤5分钟三次。
  7. 与DAPI(1:1,000)孵育10分钟,在室温下在潮湿的房间和在黑暗中
  8. 用1x PBS洗涤5分钟两次。
  9. 用荧光封固介质安装(在分析前在黑暗中储存在4℃)
  10. 在显微镜下拍照。
  11. 使用ImageJ分析测量细胞面积的图像。每个切片测量至少100个细胞,每个胚胎测量三个切片。下面是用WGA染色的紧密心肌的图像实例,其中测量相对面积。我们忽视了心肌和内皮细胞,这些细胞形成覆盖心肌细胞的单层细胞。测量心肌壁内的细胞

    图1.用荧光团偶联的小麦胚芽凝集素(WGA)染色的E15.5 WT心脏的7μm横切面

致谢

该研究由来自西班牙经济和竞争部(MINECO)的授权SAF2010-17555,RD06/0014/0038(RECAVA)和RD06/0010/1013(TERCEL)和来自J.L.d.l.P.的EU FP7-ITN215761(NotchIT) G.L.有来自MINECO的博士研究生奖学金(FPI计划,BES-2008-002904)。

参考文献

  1. Luxan,G.,Casanova,JC,Martinez-Poveda,B.,Prados,B.,D'Amato,G.,MacGrogan,D.,Gonzalez-Rajal,A.,Dobarro,D.,Torroja, Martinez,F.,Izquierdo-Garcia,JL,Fernandez-Friera,L.,Sabater-Molina,M.,Kong,YY,Pizarro,G.,Ibanez,B.,Medrano,C.,Garcia-Pavia, ,Gimeno,JR,Monserrat,L.,Jimenez-Borreguero,LJand de la Pompa,JL(2013)。 NOTCH途径调节剂 MIB1 中的突变导致左心室非压缩性心肌病。 Nat Med 19(2):193-201。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Luxán, G. and Pompa, J. L. (2013). Hypertrophy Analysis and Quantification in Embryonic Cardiomyocites. Bio-protocol 3(20): e938. DOI: 10.21769/BioProtoc.938.
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