搜索

Muscle Histology Characterization Using H&E Staining and Muscle Fiber Type Classification Using Immunofluorescence Staining
H&E染色鉴定肌肉组织学特征并通过免疫荧光染色进行肌纤维分类

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Muscle function is determined by its structure and fiber type composition. Here we describe a protocol to examine muscle histology and myofiber types using hematoxylin and eosin (H&E) and immunofluorescence staining, respectively. H&E stain nucleus in blue and cytoplasm in red, therefore allowing for morphological analyses, such as myofiber diameter, the presence of degenerated and regenerated myofibers, and adipocytes and fibrotic cells. Muscle fibers in adult skeletal muscles of rodents are classified into 4 subtypes based on the expression of myosin heavy chain proteins: Myh7 (type I fiber), Myh2 (type IIA fiber), Myh1 (type IIX fiber), Myh4 (type IIB fiber). A panel of monoclonal antibodies can be used to specifically label these muscle fiber subtypes. These protocols are commonly used in the study of muscle development, growth and regeneration (for example: Wang et al., 2015; Nie et al., 2016; Yue et al., 2016; Wang et al., 2017).

Keywords: Skeletal muscle(骨骼肌), Myofiber type(肌纤维型), Histology(组织学), H&E staining(H&E染色), Immunostaining(免疫染色)

Background

Skeletal muscle is composed of myocytes, adipocytes, fibroblasts and other cell types. Multinuclear myocytes, the main composition of skeletal muscle, are also called myofibers (muscle fibers). Investigation of muscle histology is a routine approach to study muscle function. Generally, the diameter of muscle fiber and the extent of adipocytes and fibrotic area are associated with muscle strength (Yue et al., 2016). In addition, the presence of central nucleated muscle fibers serves as a surrogate indicator of newly regenerated muscle fibers during muscle regeneration (Wang et al., 2017). Based on the differential metabolic traits and the expression of myosin heavy chain (MyHC) subtypes, myofibers are classified into four types (I, IIa, IIx and IIb). The analysis of myofiber composition is helpful for studying muscle metabolic and contractile functions (Schiaffino and Reggiani, 2011).

Materials and Reagents

  1. Clips (Universal, catalog number: UNV11240 )
  2. Kimwipes (KCWW, Kimberly-Clark, catalog number: 34155 )
  3. T-pin (Business Source, catalog number: 32351 )
  4. Blade (Crescent Manufacturing, catalog number: 7223 )
  5. Positive charged microscope slides (IMEB, catalog number: B-8255 )
  6. Cover slides (IMEB, catalog number: CG1-2450 )
  7. Adult mice
    Note: DBA/2J female mice at 6 weeks were used to show the dissection of muscles. C57BL/6 male mice at 2-month old and 1-year old were used to do stainings in this protocol, but male or female mice of other genetic backgrounds or strains, at different ages can be used.
  8. Tissue-Tek OCT compound (Sakura, catalog number: 4583 )
  9. 2-methylbutane (Fisher Scientific, catalog number: O3551-4 )
  10. Dry ice (Purdue Lilly Stores, Local dry ice supplier)
  11. Ethanol (Decon Labs, catalog number: V1001 )
  12. Xylene (Avantor® Performance Materials, Macron, catalog number: 8668 )
  13. Xylene-based mounting medium (Source Mount, catalog number: 9277722 )
  14. Primary antibodies
    Dystrophin (Abcam, catalog number: ab15277 )
    Myh2 MyHC-2A (2F7), Myh4 MyHC-2B (10F5) and Myh7 MyHC-1 (BA-F8) are from Developmental Studies Hybridoma Bank (DSHB). We are using the concentrate 0.1 ml products
  15. Phosphate-buffered saline (PBS) (pH 7.4) (Sigma-Aldrich, catalog number: P3813 )
  16. Mounting medium for immunostaining (Agilent Technologies, catalog number: S3023 )
  17. Nail polish (Crystal clear) (Sally Hansen, catalog number: 004677963 )
  18. Secondary antibodies
    Goat anti-Mouse IgG1, Alexa Fluor 568 (Thermo Fisher Scientific, Invitrogen, catalog number: A-21124 )
    Goat anti-Mouse IgG1, Alexa Fluor 488 (Thermo Fisher Scientific, Invitrogen, catalog number: A-21121 )
    Goat anti-Mouse IgG2b, Alexa Fluor 568 (Thermo Fisher Scientific, Invitrogen, catalog number: A-21144 )
    Goat anti-Mouse IgG2b, Alexa Fluor 647 (Thermo Fisher Scientific, Invitrogen, catalog number: A-21242 )
    Goat anti-Mouse IgM, Alexa Fluor 488 (Thermo Fisher Scientific, Invitrogen, catalog number: A-21042 )
    Goat anti-Rabbit IgG (H+L), Alexa Fluor 647 (Thermo Fisher Scientific, Invitrogen, catalog number: A-21245 )
  19. Hematoxylin (Sigma-Aldrich, catalog number: H9627 )
  20. Potassium alum (Sigma-Aldrich, catalog number: 237086 )
  21. Sodium iodate (Sigma-Aldrich, catalog number: S4007 )
  22. Glacial acetic acid (Avantor® Performance Materials, Macron, catalog number: V193 )
  23. Glycerol (Avantor® Performance Materials, Macron, catalog number: 5092 )
  24. Eosin Y (Santa Cruz Biotechnology, catalog number: sc-203734 )
  25. Goat serum (Jackson ImmunoResearch, catalog number: 005-000-121 )
  26. Bovine serum albumin (BSA) (Gemini Bio-Products, catalog number: 700-105P )
  27. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  28. Sodium azide (Fisher Scientific, catalog number: S2271 )
  29. Hematoxylin solution (see Recipes)
  30. Eosin Y solution (see Recipes)
  31. Blocking buffer (see Recipes)

Equipment

  1. Embedding molds (Structure Probe, SPI Supplies, catalog number: 2449M-AB )
  2. Cold-resistant beaker (Sigma-Aldrich, catalog number: Z155519 )
  3. -80 °C freezer (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM 8600 Series )
  4. Leica CM1850 cryostat (Leica Biosystems, model: CM1850 )
  5. Staining jar (BRAND, catalog number: 472800 )
  6. Fume hood (Fisher Scientific, model: Fisher Hamilton SafeAire )
  7. 14.0 MP digital USB microscope camera (OMAX Microscope, catalog number: A35140U3 )
  8. Leica DMI 6000B fluorescent microscope (Leica Microsystems, model: DMI 6000 B )
  9. Lumen 200 fluorescence illumination system (Prior Scientific, model: Lumen 200 )
  10. Coolsnap HQ CCD camera (Photometrics, model: Coolsnap HQ )
  11. Liquid blocker (Ted Pella, catalog number: 22309 )
  12. Dissecting scissors, 10 cm, straight (WPI, catalog number: 14393-G )
  13. Vannas scissors, 8 cm, straight (WPI, catalog number: 14003-G )
  14. Tweezer, 4.25”, straight (Excelta, catalog number: 5-S-SE )
  15. Tweezer, 4.75”, straight (Excelta, catalog number: 3-S-SE )

Software

  1. Photoshop software (e.g., Photoshop CC)

Procedure

  1. Dissection of muscles
    1. Tools used to dissect muscles of mice are shown in Figure 1A. Video 1 shows how to dissect muscles from a mouse.
    2. Position the mouse supine on a dissection board (Styrofoam board) and secure the leg with a T-pin (Figure 1B).
    3. Peel off the leg skin to expose the hind limb muscles. Expose tendons of tibialis anterior (TA) muscle and extensor digitorum longus (EDL) muscle (Figure 1B).
    4. Gently remove the fascia covering the TA muscle.
    5. Cut the distal TA tendon and use it to peel off the TA muscle. Carefully remove the TA muscle at its proximal attachment (Figure 1C).
    6. After removing the TA muscle, cut distal EDL muscle and peel off the EDL muscle. Carefully cut the proximal EDL tendon and remove the EDL muscle. To dissect soleus muscle (SOL), position the mouse prone on a dissection board and secure the leg with a T-pin (Figure 1D).
    7. Cut the tendon of gastrocnemius (GA) muscle and use it to peel off the GA muscle till the exposure of a SOL tendon (Figure 1E).
    8. Cut the SOL tendon and use it to peel off the SOL muscle (Figure 1F).

    Figure 1. Procedure of dissection muscles from a mouse. A. Tools used to dissect muscles; B-F. Key steps for muscle dissection; G. Illustration of how muscles are placed in embedding molds with O.C.T. compound. SOL is in the left mold and TA is in the right mold.


    Video 1. Dissection of skeletal muscles from a mouse

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


  2. Embedding of muscle
    1. After dissection, skeletal muscles (e.g., tibialis anterior, soleus, extensor digitorum longus) are embedded in embedding molds (e.g., SPI Supplies) with minimum amount of Tissue-Tek O.C.T. compound possible to cover the muscles (Figure 1G).
    2. Place a cold-resistant beaker of 2-methylbutane into a slurry of dry ice and ethanol (pure), which allows fast cooling to -78 °C. When the correct temperature is attained, no bubble will appear after putting a piece of dry ice into the 2-methylbutane.
    3. Freeze the embedded muscle by placing it into the cooled 2-methylbutane for 5 min and then transfer the muscle sample to a -80 °C freezer for storage.
  3. Cryosectioning
    1. Before cryosectioning, the cryostat with the blade is pre-cooled to -22 ± 2 °C. Samples are placed in cryostat for at least 20 min for thermal equilibration. Attach the sample on the round metallic holders of the cryostat with Tissue-Tek O.C.T.
    2. Make 10 µm-thick sections and collect them on room temperature positive charged microscope slides and then store the slides in a slide box at a -80 °C freezer. These slides can be processed further for H&E staining or immunostaining.
  4. H&E staining
    1. Bring the slides from the -80 °C freezer to room temperature.
    2. Incubate the slides with hematoxylin solution in a staining jar for 10 min to stain the nuclei.
    3. Transfer the slides to a staining jar with running water (tap water is fine) till the water is clear.
    4. Transfer the slides to a staining jar with Eosin solution for 3 min.
    5. Successively transfer the slides into staining jars with 70% ethanol for 20 sec, 90% ethanol for 20 sec, 100% ethanol for 1 min and xylene for 3 min.
    6. Take out slides from xylene and place the slides in a fume hood till the slides are dry.
    7. Mount the slides with xylene-based mounting media and cover with cover slides. Clips are used to press the slides to squeeze bubbles.
    8. Store the slides at room temperature.
    9. Hematoxylin and Eosin-stained images were captured with a 14.0 MP digital microscope camera which is attached via a c-mount to the side port of a Leica DMI 6000B microscope.
  5. Immunostaining with MyHC antibodies
    1. Bring the slides from the -80 °C freezer to room temperature and surround the sections with a liquid blocker (Ted Pella) to limit the usage of the incubation solutions.
    2. Place the slides in a wet chamber (Note 5) and block the sections with blocking buffer for at least 30 min at room temperature. For the immunostaining with MyHC antibodies from DSHB, do not fix samples or sections with fixative solution (this is specifically for the MyHC antibodies).
    3. Dilute primary antibodies with blocking buffer. Myh2, Myh4 and Myh7 antibodies are diluted at a ratio 1:300. Dystrophin antibody is diluted at a ratio 1:1,000. Remove blocking buffer from the sections and add diluted primary antibodies on sections. Place the slides in a wet chamber overnight at 4 °C.
    4. Dilute secondary antibodies with PBS at a ratio 1:1,000. Incubate secondary antibodies with sections for 30 min to 1 h at room temperature. Slides are placed in a wet chamber.
    5. Wash the slides with PBS for 5 times. Before mounting the slides, remove PBS as much as possible, but keep sections wet.
    6. Mount the slides with immunostaining mounting media and cover with cover slides. Clips are used to press the slides to squeeze bubbles. Seal the slides with clear nail polish.
    7. Store the slides at 4 °C.
    8. Fluorescent images were captured using the Leica DMI 6000B fluorescent microscope.

Data analysis

  1. H&E staining of Tibialis anterior muscle sections
    Cardiotoxin (CTX) was injected into tibialis anterior (TA) muscles of 1-year old male mice to induce muscle damage which was followed by muscle regeneration. Two weeks after CTX injection, TA muscles were processed for cryosection and H&E staining (Figure 2). Most myofibers had central nuclei, indicative of muscle regeneration (Figure 2). Adipocytes and degenerated myofibers were found in TA muscle sections (Figure 2).


    Figure 2. H&E staining of regenerated TA muscles isolated from 1-year old mice. Re/degenerated myofibers and adipocytes are indicated by different arrows. Scale bars = 100 µm.


  2. Immunostaining of myofiber types
    SOL muscles and TA muscles of 2-month old male mice were processed for immunostaining of myosin heavy chain (MyHC). For SOL muscles, we used Myh2, Myh7 and Dystrophin primary antibodies to stain Type IIa, Type I myofibers and sarcolemma, respectively. Goat anti-Mouse IgG1-488, Goat anti-Mouse IgG2b-568 and Goat anti-Rabbit IgG-647 secondary antibodies were used to distinguish Myh2, Myh7 and Dystrophin primary antibodies, respectively. SOL muscle is ‘slow’ muscle which contains Type I (Red), Type IIa (Green) and Type IIx (Black) myofibers (Figure 3).


    Figure 3. Immunostaining of myofibers in SOL muscles of 2-month old male mice. Myh2, Myh7 and Dystrophin primary antibodies were used to stain Type IIa (Green), Type I (Red) myofibers and sarcolemma (Blue), respectively. Unlabeled myofibers (Black) are presumed to be Type IIx myofibers. One Type IIx myofiber is indicated by the arrow. Scale bar = 200 µm.

    For TA muscles, we used Myh2, Myh4 and Myh7 primary antibodies to stain Type IIa, Type IIb and Type I myofibers, respectively. Goat anti-Mouse IgG1-568, Goat anti-Mouse IgM-488, and Goat anti-Mouse IgG2b-647 secondary antibodies were used to distinguish Myh2, Myh4 and Myh7 primary antibodies, respectively. TA muscle is ‘fast’ muscle which is dominated by Type IIa (Red), Type IIx (Black) and Type IIb (Green) myofibers (Figure 4). Few Type I myofibers exist in TA muscles (Figure 4).


    Figure 4. Immunostaining of myofibers in TA muscles of 2-month old male mice. Myh2, Myh4 and Myh7 primary antibodies were used to stain Type IIa (Red), Type IIb (Green) and Type I (Blue) myofibers, respectively. Unlabeled myofibers (Black) are presumed to be Type IIx myofibers. One Type IIx myofiber is indicated by the arrow. Scale bar = 500 µm.

    The numbers of myofibers are counted with Photoshop software (we use Photoshop CC, but any version of Photoshop containing the Count Tool works). For example, when counting different types of myofibers in a SOL muscle section, first, open image in Photoshop CC (Figure 5A). Next, choose Image > Analysis > Count Tool (Figure 5A). Next, choose Red and Blue channels (bottom right corner) to show Type I myofibers and sarcolemma (Figure 5B). A number will appear in a myofiber when you click the myofiber. Total number can be found in the Counts bar (top left corner, oval labeled, Figure 5B). After counting Type I myofibers, choose Green and Blue channels to count Type IIa myofibers. At last, choose Red, Green and Blue channels, myofibers without Red or Green labelling are Type IIx myofibers.


    Figure 5. Illustration of myofiber counting in Photoshop CC. A. Open Count Tool in Photoshop CC; B. Choose a type of myofiber and count the number by clicking the myofiber. Total number is shown in the Count bar (oval highlighted).

Notes

  1. Thermal equilibration is indispensable before cryosectioning.
  2. The H&E staining protocol is generated based on our daily practice. It is not necessary to fix the sections before the hematoxylin staining. In addition, the ‘Blueing’ procedure is not required after the hematoxylin staining.
  3. In the immunostaining using MyHC antibodies from DSHB, avoid fixation of the sections. Fixation disrupts the binding of antibody to myosin heavy chain.
  4. The Abcam Dystrophin antibody works well in fixed and non-fixed sections.
  5. The wet chamber is made by placing several wet papers in a slide box (Figure 6). Place a slide in a wet chamber as it is shown in Figure 6.


    Figure 6. Setup of a wet chamber and placement of a slide in a wet chamber

Recipes

  1. Hematoxylin solution
    6 g hematoxylin
    100 ml ethanol
    150 g potassium alum
    2,000 ml double-distilled H2O
    1.2 g sodium iodate
    120 ml glacial acetic acid
    900 ml glycerol
    Dissolve the hematoxylin in ethanol, and the potassium alum in distilled water. After that, mix them with glycerol, and add sodium iodate and glacial acetic acid at last. Store hematoxylin solution at room temperature
  2. Eosin Y solution
    2 g Eosin Y
    40 ml double-distilled H2O
    760 ml 95% ethanol
    4 ml glacial acetic acid
    Dissolve Eosin Y in double-distilled H2O. Then add 95% ethanol, and mix. While working in a fume hood, add glacial acetic acid and mix well. Store covered at room temperature
  3. Blocking buffer
    5% goat serum
    2% BSA
    0.2% Triton X-100
    0.1% sodium azide
    200 ml blocking buffer is prepared with PBS containing 10 ml goat serum, 4 g BSA, 4 ml 10% Triton X-100 and 0.2 g sodium azide. Aliquot and store at -20 °C

Acknowledgments

This work was supported by a grant from the US National Institutes of Health (R01AR071649 to S.K.).

References

  1. Nie, Y., Sato, Y., Wang, C., Yue, F., Kuang, S. and Gavin, T. P. (2016). Impaired exercise tolerance, mitochondrial biogenesis, and muscle fiber maintenance in miR-133a-deficient mice. FASEB J 30(11): 3745-3758.
  2. Schiaffino, S. and Reggiani, C. (2011). Fiber types in mammalian skeletal muscles. Physiol Rev 91(4): 1447-1531.
  3. Wang, C., Wang, M., Arrington, J., Shan, T., Yue, F., Nie, Y., Tao, W. A. and Kuang, S. (2017). Ascl2 inhibits myogenesis by antagonizing the transcriptional activity of myogenic regulatory factors. Development 144(2): 235-247.
  4. Wang, J. H., Wang, Q. J., Wang, C., Reinholt, B., Grant, A. L., Gerrard, D. E. and Kuang, S. (2015). Heterogeneous activation of a slow myosin gene in proliferating myoblasts and differentiated single myofibers. Dev Biol 402(1): 72-80.
  5. Yue, F., Bi, P., Wang, C., Li, J., Liu, X. and Kuang, S. (2016). Conditional loss of Pten in myogenic progenitors leads to postnatal skeletal muscle hypertrophy but age-dependent exhaustion of satellite cells. Cell Rep 17(9): 2340-2353.

简介

肌肉功能由其结构和纤维类型组成决定。 这里我们描述一个协议,分别检查使用苏木精和伊红(H&E)和免疫荧光染色的肌肉组织学和肌纤维类型。 H&E在蓝色和细胞质红色染色核,因此允许形态学分析,如肌纤维直径,退化和再生肌纤维的存在,以及脂肪细胞和纤维化细胞。 基于肌球蛋白重链蛋白Myh7(I型纤维),Myh2(IIA型纤维),Myh1(IIX型纤维),Myh4(IIB型纤维)的表达,将啮齿动物成年骨骼肌肌肉纤维分为4种亚型:。 一组单克隆抗体可用于特异性标记这些肌纤维亚型。 这些方案通常用于肌肉发育,生长和再生的研究(例如:Wang等,2015; Nie等,2016; Yue et al。,2016; Wang et al。,2017)。
【背景】骨骼肌由肌细胞,脂肪细胞,成纤维细胞和其他细胞类型组成。 多核心肌细胞,骨骼肌的主要组成,也称为肌纤维(肌纤维)。 肌肉组织学研究是肌肉功能研究的常规方法。 一般来说,肌纤维的直径和脂肪细胞的程度和纤维化面积与肌肉力量有关(Yue et al。,2016)。 此外,中心有核肌纤维的存在作为肌肉再生期间新再生肌纤维的替代指标(Wang等,2017)。 基于差异代谢性状和肌球蛋白重链(MyHC)亚型的表达,肌纤维分为四种类型(I,IIa,IIx和IIb)。 肌纤维组成的分析有助于研究肌肉代谢和收缩功能(Schiaffino和Reggiani,2011)。

关键字:骨骼肌, 肌纤维型, 组织学, H&E染色, 免疫染色

材料和试剂

  1. 剪辑(通用,目录号:UNV11240)
  2. Kimwipes(KCWW,Kimberly-Clark,目录号:34155)
  3. T-pin(业务来源,目录号:32351)
  4. 刀片(新月制造,目录号:7223)
  5. 带正电的显微镜幻灯片(IMEB,目录号:B-8255)
  6. 封面幻灯片(IMEB,目录号:CG1-2450)
  7. 成年老鼠
    注意:DBA/2J雌性小鼠在6周时用于显示肌肉的解剖。使用2月龄和1岁的C57BL/6雄性小鼠在本方案中进行染色,但可以使用不同年龄的其他遗传背景或菌株的雄性或雌性小鼠。 >
  8. Tissue-Tek OCT化合物(Sakura,目录号:4583)
  9. 2-甲基丁烷(Fisher Scientific,目录号:O3551-4)
  10. 干冰(Purdue Lilly Stores,当地干冰供应商)
  11. 乙醇(Decon Labs,目录号:V1001)
  12. 二甲苯(Avantor ®性能材料,Macron,目录号:8668)
  13. 二甲苯基安装介质(源安装,目录号:9277722)
  14. 初级抗体
    肌营养不良蛋白(Abcam,目录号:ab15277)
    Myh2 MyHC-2A(2F7),Myh4 MyHC-2B(10F5)和Myh7 MyHC-1(BA-F8)来自发育研究杂交瘤银行(DSHB)。我们正在使用浓缩液0.1 ml产品
  15. 磷酸盐缓冲盐水(PBS)(pH 7.4)(Sigma-Aldrich,目录号:P3813)
  16. 用于免疫染色的安装介质(Agilent Technologies,目录号:S3023)
  17. 指甲油(水晶透明)(Sally Hansen,目录号:004677963)
  18. 二次抗体
    山羊抗小鼠IgG1,Alexa Fluor 568(Thermo Fisher Scientific,Invitrogen,目录号:A-21124)
    山羊抗小鼠IgG1,Alexa Fluor 488(Thermo Fisher Scientific,Invitrogen,目录号:A-21121)
    山羊抗小鼠IgG2b,Alexa Fluor 568(Thermo Fisher Scientific,Invitrogen,目录号:A-21144)
    山羊抗小鼠IgG2b,Alexa Fluor 647(Thermo Fisher Scientific,Invitrogen,目录号:A-21242)
    山羊抗小鼠IgM,Alexa Fluor 488(Thermo Fisher Scientific,Invitrogen,目录号:A-21042)
    山羊抗兔IgG(H + L),Alexa Fluor 647(Thermo Fisher Scientific,Invitrogen,目录号:A-21245)
  19. 苏木精(Sigma-Aldrich,目录号:H9627)
  20. 钾明矾(Sigma-Aldrich,目录号:237086)
  21. 碘酸钠(Sigma-Aldrich,目录号:S4007)
  22. 冰醋酸(Avantor 性能材料,Macron,目录号:V193)
  23. 甘油(Avantor ®性能材料,Macron,目录号:5092)
  24. Eosin Y(Santa Cruz Biotechnology,目录号:sc-203734)
  25. 山羊血清(Jackson ImmunoResearch,目录号:005-000-121)
  26. 牛血清白蛋白(BSA)(Gemini Bio-Products,目录号:700-105P)
  27. Triton X-100(Sigma-Aldrich,目录号:X100)
  28. 叠氮化钠(Fisher Scientific,目录号:S2271)
  29. 苏木精溶液(参见食谱)
  30. 曙红Y溶液(见配方)
  31. 阻塞缓冲区(见配方)

设备

  1. 嵌入模具(结构探头,SPI耗材,目录号:2449M-AB)
  2. 耐寒烧杯(Sigma-Aldrich,目录号:Z155519)
  3. -80°C冰箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma TM 8600系列)
  4. Leica CM1850低温恒温器(Leica Biosystems,型号:CM1850)
  5. 染色罐(BRAND,目录号:472800)
  6. 通风柜(Fisher Scientific,型号:Fisher Hamilton SafeAire)
  7. 14.0 MP数码USB显微镜相机(OMAX显微镜,目录号:A35140U3)
  8. 徕卡DMI 6000B荧光显微镜(Leica Microsystems,型号:DMI 6000 B)
  9. Lumen 200荧光照明系统(Prior Scientific,型号:Lumen 200)
  10. Coolsnap HQ CCD相机(Photometrics,型号:Coolsnap HQ)
  11. 液体阻滞剂(Ted Pella,目录号:22309)
  12. 解剖剪刀,10厘米直(WPI,目录号:14393-G)
  13. Vannas剪刀,8厘米,直(WPI,目录号:14003-G)
  14. 镊子,4.25",直(Excelta,目录号:5-S-SE)
  15. 镊子,4.75",直(Excelta,目录号:3-S-SE)

软件

  1. Photoshop软件(例如,,Photoshop CC)

程序

  1. 解剖肌肉
    1. 用于解剖小鼠肌肉的工具如图1A所示。视频1显示如何解剖小鼠的肌肉。
    2. 将鼠标仰卧在解剖板(聚苯乙烯泡沫板)上,并用T形针固定腿部(图1B)。
    3. 剥离腿部皮肤,以暴露后肢肌肉。暴露肌腱前肌(TA)肌肉和伸肌腱长肌(EDL)肌肉(图1B)。
    4. 轻轻取出覆盖TA肌肉的筋膜。
    5. 切割远端TA肌腱并用它剥离TA肌肉。在其附近小心地移除TA肌肉(图1C)。
    6. 去除TA肌肉后,切开远端的EDL肌肉并剥离EDL肌肉。仔细切开近端EDL肌腱并取出EDL肌肉。为了解剖比目鱼肌(SOL),将鼠标放置在解剖板上并用T形针固定腿(图1D)。
    7. 切割腓肠肌(GA)肌肉的肌腱,并用它剥离GA肌肉,直到暴露于肌腱(图1E)。
    8. 切割SOL肌腱并用它剥离SOL肌肉(图1F)。

    图1.从小鼠中解剖肌肉的程序。 A.用于解剖肌肉的工具; B-F。肌肉解剖的关键步骤G.如何将肌肉放置在用O.C.T.嵌入模具中。复合。 SOL在左模,TA位于右模。


  2. 嵌入肌肉
    1. 在解剖后,将骨骼肌(例如,胫前肌,比目鱼肌,伸肌腱)以最小量的Tissue-Tek O.C.T.嵌入嵌入模具(例如,SPI Supplies)中。复合可能覆盖肌肉(图1G)。
    2. 将2-甲基丁烷的耐寒烧杯放入干冰和乙醇(纯)的浆液中,使其能够快速冷却至-78℃。当达到正确的温度时,将一块干冰放入2-甲基丁烷后不会出现气泡。
    3. 将嵌入的肌肉放入冷却的2-甲基丁烷中5分钟,然后将肌肉样品转移到-80°C的冷冻箱中,以将其保存下来。
  3. 冷冻切片
    1. 在冷冻切片之前,将具有刀片的低温恒温器预冷至-22±2℃。将样品置于低温恒温器中至少20分钟以进行热平衡。使用Tissue-Tek O.C.T.将样品安装在低温恒温器的圆形金属支架上。
    2. 制成10μm厚的切片,并将其收集在室温的带正电荷的显微镜载玻片上,然后将载玻片存放在-80℃冷冻箱的载玻片盒中。这些载玻片可进一步处理H& E染色或免疫染色。
  4. H& E染色
    1. 将幻灯片从-80°C冰箱送至室温。
    2. 使用苏木精溶液在染色瓶中孵育载玻片10分钟以染色细胞核
    3. 将载玻片转移到带有自来水(自来水很好)的染色罐中,直到水清除
    4. 将幻灯片转移到含有曙红溶液的染色瓶中3分钟
    5. 连续地将载玻片转移到具有70%乙醇的染色瓶中20秒,90%乙醇20秒,100%乙醇1分钟和二甲苯3分钟。
    6. 从二甲苯取出幻灯片,将幻灯片放在通风橱中,直到幻灯片干燥。
    7. 用二甲苯安装介质和盖子盖子滑块安装幻灯片。剪辑用于按压幻灯片以挤压气泡。
    8. 在室温下存放载玻片。
    9. 苏木精和曙红染色的图像用14.0 MP数字显微镜相机拍摄,该相机通过c-mount连接到Leica DMI 6000B显微镜的侧面端口。
  5. 用MyHC抗体免疫染色
    1. 将幻灯片从-80°C冷冻箱中放置到室温,并用液体阻塞剂(Ted Pella)围住部分,以限制孵育溶液的使用。
    2. 将载玻片置于湿室中(注5),并在室温下用封闭缓冲液封闭至少30分钟。对于使用来自DSHB的MyHC抗体进行免疫染色,不要用固定液(特别是MyHC抗体)固定样品或切片。
    3. 用封闭缓冲液稀释一抗。 Myh2,Myh4和Myh7抗体以1:300的比例稀释。肌营养不良蛋白抗体以1:1,000的比例稀释。从切片中除去封闭缓冲液,并在切片上加入稀释的一抗。将载玻片置于湿室中4℃过夜。
    4. 用PBS以1:1,000的比例稀释二次抗体。在室温下将二级抗体与切片孵育30分钟至1小时。幻灯片放置在湿室中。
    5. 用PBS洗涤载玻片5次。安装幻灯片之前,请尽可能多地移除PBS,但请保持部分潮湿。
    6. 用免疫染色安装介质和盖子盖上幻灯片安装幻灯片。剪辑用于压缩幻灯片以挤压气泡。用透明指甲油密封幻灯片。
    7. 将幻灯片存放在4°C。
    8. 使用Leica DMI 6000B荧光显微镜拍摄荧光图像。

    数据分析

    1. 胫骨前肌切片的H& E染色 将心脏毒素(CTX)注射到1岁雄性小鼠的胫前肌(TA)肌肉中以诱导肌肉损伤,随后进行肌肉再生。 CTX注射两周后,将TA肌肉加工用于冷冻切片和H& E染色(图2)。大多数肌纤维具有中枢核,指示肌肉再生(图2)。在TA肌肉部分发现脂肪细胞和退化肌纤维(图2)

      图2.从1岁小鼠分离的再生TA肌肉的H& E染色 Re /变性肌纤维和脂肪细胞用不同的箭头表示。比例尺=100μm

    2. 免疫染色的肌纤维类型
      处理2个月大的雄性小鼠的SOL肌肉和TA肌肉用于肌球蛋白重链(MyHC)的免疫染色。对于SOL肌肉,我们使用Myh2,Myh7和Dystrophin一抗分别染色IIa型,I型肌纤维和肌膜。山羊抗小鼠IgG1-488,山羊抗小鼠IgG2b-568和山羊抗兔IgG-647二抗分别用于区分Myh2,Myh7和肌营养不良蛋白一抗。 SOL肌肉是含有I型(红色),IIa型(绿色)和IIx型(黑色)肌纤维(图3)的"慢肌"。

      图3.在2月龄雄性小鼠的SOL肌肉中肌纤维的免疫染色 Myh2,Myh7和肌营养不良蛋白一抗被用于染色IIa型(绿色),I型(红色)肌纤维和肌膜蓝色)。未标记的肌纤维(黑色)被推定为IIx型肌纤维。一种IIx型肌纤维用箭头表示。比例尺= 200μm
      对于TA肌肉,我们使用Myh2,Myh4和Myh7一抗来分别染色IIa型,IIb型和I型肌纤维。山羊抗小鼠IgG1-568,山羊抗小鼠IgM-488和山羊抗小鼠IgG2b-647二抗分别用于区分Myh2,Myh4和Myh7一抗。 TA肌肉是IIa型(红色),IIx型(黑色)和IIb型(绿色)肌纤维主导的"快速"肌肉(图4)。 TA肌肉中存在少量I型肌纤维(图4)

      图4.在2个月大的雄性小鼠的TA肌肉中肌纤维的免疫染色。 Myh2,Myh4和Myh7一抗被用于分别染色IIa型(红色),IIb型(绿色)和I型(蓝色)肌纤维。未标记的肌纤维(黑色)被推定为IIx型肌纤维。一种IIx型肌纤维用箭头表示。比例尺= 500μm。

      使用Photoshop软件计算肌纤维数量(我们使用Photoshop CC,但包含计数工具的任何版本的Photoshop)。例如,当在SOL肌肉部分中计数不同类型的肌纤维时,首先,在Photoshop CC中打开图像(图5A)。接下来,选择图像>分析>计数工具(图5A)。接下来,选择红色和蓝色通道(右下角)显示I型肌纤维和肌膜(图5B)。当您点击肌纤维时,一个数字将出现在肌纤维中。总数可以在"计数"栏中找到(左上角,椭圆标记,图5B)。在计算I型肌纤维后,选择绿色和蓝色通道计数IIa型肌纤维。最后,选择红色,绿色和蓝色通道,没有红色或绿色标签的肌纤维是IIx型肌纤维。


      图5. Photoshop CC中肌肉计数的图示。 A.在Photoshop CC中打开计数工具; B.选择一种肌纤维,然后点击肌纤维来计数。总数显示在计数栏中(椭圆形突出显示)。

    笔记

    1. 热平衡在冷冻切除之前是不可或缺的
    2. H& E染色方案是根据我们的日常实践生成的。在苏木精染色前不需要固定切片。此外,在苏木精染色后不需要"蓝化"程序。
    3. 在使用来自DSHB的MyHC抗体的免疫染色中,避免固定部分。固定会破坏抗体与肌球蛋白重链的结合
    4. Abcam抗肌萎缩蛋白抗体在固定和非固定切片中工作良好。
    5. 湿室是通过将几张湿纸放在滑动盒中制成的(图6)。将幻灯片放在湿室中,如图6所示。


      图6.湿室的设置和在湿室中放置滑块

    食谱

    1. 苏木精溶液
      6克苏木精
      100ml乙醇
      150克钾明矾
      2,000ml双蒸H 2 O 2/
      1.2克碘酸钠
      120 ml冰醋酸
      900毫升甘油
      将苏木精溶于乙醇中,并在蒸馏水中溶解钾明矾。之后,将其与甘油混合,最后加入碘酸钠和冰醋酸。在室温下储存苏木精溶液
    2. Eosin Y解决方案
      2 g Eosin Y
      40ml双蒸H 2 O 2/
      760 ml 95%乙醇 4毫升冰醋酸
      将曙红Y溶于双蒸H 2 O。然后加入95%乙醇,混匀。在通风橱中工作时,加入冰醋酸并充分混合。商店在室温下覆盖
    3. 阻塞缓冲区
      5%山羊血清
      2%BSA
      0.2%Triton X-100
      0.1%叠氮化钠
      用含有10ml山羊血清,4g BSA,4ml 10%Triton X-100和0.2g叠氮化钠的PBS制备200ml封闭缓冲液。等分并储存于-20°C

    致谢

    这项工作得到了美国国家卫生研究院(R01AR071649至S.K.)的资助。

    参考

    1. Nie,Y.,Sato,Y.,Wang,C.,Yue,F.,Kuang,S.and Gavin,TP(2016)。  在miR-133a缺陷小鼠中受损的运动耐力,线粒体生物发生和肌肉纤维维持。 FASEB J 30(11):3745-3758。
    2. Schiaffino,S.和Reggiani,C.(2011)。哺乳动物骨骼肌中的纤维类型。生物化学版91(4):1447-1531。
    3. Wang,C.,Wang,M.,Arrington,J.,Shan,T.,Yue,F.,Nie,Y.,Tao,WA and Kuang,S。(2017)。< a class = -insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27993983"target ="_ blank"> Ascl2通过拮抗肌原性调节因子的转录活性来抑制肌发生。发展 144(2):235-247。
    4. Wang,JH,Wang,QJ,Wang,C.,Reinholt,B.,Grant,AL,Gerrard,DE and Kuang,S。(2015)。  增殖成肌细胞和分化单一肌纤维中慢肌球蛋白基因的异质活化。 Dev Biol 402(1):72-80。
    5. Yue,F.,Bi,P.,Wang,C.,Li,J.,Liu,X.and Kuang,S。(2016)。  在肌原性祖细胞中有条件损失Pten 导致产后骨骼肌肥大,但是卫星细胞的年龄依赖性耗尽。 Cell Rep 17(9):2340-2353。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017,  Wang et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Wang, C., Yue, F. and Kuang, S. (2017). Muscle Histology Characterization Using H&E Staining and Muscle Fiber Type Classification Using Immunofluorescence Staining. Bio-protocol 7(10): e2279. DOI: 10.21769/BioProtoc.2279.
  2. Bi, P., Yue, F., Sato, Y., Wirbisky, S., Liu, W., Shan, T., Wen, Y., Zhou, D., Freeman, J. and Kuang, S. (2016). Stage-specific effects of Notch activation during skeletal myogenesis. Elife 5.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。