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Lung Section Staining and Microscopy
肺切片染色和显微镜观察   

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参见作者原研究论文

本实验方案简略版
Mucosal Immunology
May 2016

 

Abstract

Our protocol describes immunofluorescent staining, hematoxylin and eosin staining and Masson’s trichrome staining on lung sections.

Keywords: Immunofluorescent staining (免疫荧光染色), Hematoxylin and eosin staining (苏木精和伊红染色), Masson’s trichrome staining (Masson三色染色), Frozen tissue sections (冷冻组织切片), Paraffin-embedded tissue sections (石蜡包埋组织切片), Antibodies (抗体)

Background

The primary function of the lungs is gas exchange. The lungs are composed of various specialized cells and tissues, including the bronchi, the bronchioles and the pulmonary alveoli to facilitate gas exchange. To study lung development or lung diseases in mouse models, the lungs can be removed from mice and either frozen and embedded in optimal cutting temperature (OCT) compound or chemically preserved and embedded in paraffin. To preserve lung tissue architecture, we filled the lung with 1 ml of OCT compound for preparing frozen sections, or 10% buffered formalin for paraffin sections (Zhou et al., 2016). Lung sections are then sliced from frozen or paraffin-embedded lungs and mounted onto slides for preparation of staining. We used frozen tissue sections for antibody-based immunofluorescent staining and used paraffin-embedded sections for hematoxylin and eosin (H&E) staining or Masson’s trichrome staining. Frozen sections are quicker to prepare for immunofluorescent staining and most antibodies work well on frozen sections. Paraffin sections can also be used for immunofluorescent staining, but they require deparaffinization, rehydration and antigen retrieval. Some antibodies do not work well on paraffin sections even after antigen retrieval. However, paraffin samples can be stored at room temperate for very long periods and can be easily cut into very thin sections. Paraffin sections preserve better tissue morphology than frozen sections, so they are better for H&E or trichrome staining.

Immunofluorescent staining is a type of immunohistochemistry that makes use of fluorophores to visualize the location of the antibodies that specifically bind to their target proteins. H&E staining is the most widely used stain in histology and medical diagnosis. This staining method involves application of hemalum and eosin Y that color cell nuclei blue and cytoplasm pink to red. Masson’s trichrome staining is a three-color staining that is used for detecting collagen fibers in tissues. The staining produces blue collagen, dark brown to black cell nuclei and red background.

Materials and Reagents

  1. Fisherbrand Superfrost Plus microscope slides (Fisher Scientific, catalog number: 12-550-15 )
  2. Disposable mold
  3. Coverslip slides
  4. C57BL/6 mice
  5. Tissue-Tek OCT compound (SAKURA FINETEK, catalog number: 4583 )
  6. Formaldehyde, 37-40% ACS (Newcomer Supply, catalog number: 1089 )
  7. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  8. Phosphate buffered saline (PBS, pH 7.4) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  9. Serum from secondary antibody’s host
  10. Primary antibodies: Rabbit anti α-smooth muscle actin (α-SMA) antibody (Abcam, catalog number: ab5694 )
  11. Secondary reagents: Texas Red conjugated goat anti-rabbit IgG antibody (Abcam, catalog number: ab6719 )
  12. Prolong® Gold anti-fade reagent (Cell Signaling Technology, catalog number: 9071 ), or with DAPI (Cell Signaling Technology, catalog number: 8961 )
  13. Paraffin
  14. Ethyl alcohol (Newcomer Supply, catalog number: 10841 )
  15. Xylene (Newcomer Supply, catalog number: 1446 )
  16. S-mounting medium (Newcomer Supply, catalog number: 6750 )
  17. Bouin Fluid (Newcomer Supply, catalog number: 1020 )
  18. Biebrich scarlet-acid fuchsin solution (Sigma-Aldrich, catalog number: HT151 )
  19. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A3912 )
  20. Fetal bovine serum (FBS) (GE Healthcare, HycloneTM, catalog number: SH30396.03 )
  21. Eosin Y (Sigma-Aldrich, catalog number: E4009 )
  22. Acetic acid, glacial (Sigma-Aldrich, catalog number: A6283 )
  23. Aluminum potassium sulfate (Sigma-Aldrich, catalog number: 237086 )
  24. Hematoxylin (Sigma-Aldrich, catalog number: H3136 )
  25. Sodium iodate (Sigma-Aldrich, catalog number: S4007 )
  26. Citric acid (Sigma-Aldrich, catalog number: 251275 )
  27. Ferric chloride (Sigma-Aldrich, catalog number: 157740 )
  28. Hydrochloric acid (Sigma-Aldrich, catalog number: 320331 )
  29. Phosphomolybdic acid solution (Sigma-Aldrich, catalog number: HT153 )
  30. Phosphotungstic acid solution (Sigma-Aldrich, catalog number: HT152 )
  31. Aniline blue (Sigma-Aldrich, catalog number: 415049 )
  32. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  33. Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S8282 )
  34. Blocking buffer (see Recipes)
  35. Eosin Y solution (see Recipes)
  36. Eosin Y working solution (0.25%) (see Recipes)
  37. Mayer’s hematoxylin solution (see Recipes)
  38. Weigert’s iron hematoxylin solution (see Recipes)
  39. Phosphomolybdic-phosphotungstic acid solution (see Recipes)
  40. Aniline blue solution (see Recipes)

Equipment

  1. Cryostat microtome
  2. -70 °C freezer
  3. Fluorescence microscope or a confocal microscope
  4. Staining jar and slide rack, for example EasyDip Slide Staining System (Newcomer Supply, catalog number: 5300KIT )
  5. Optical microscope

Procedure

  1. Immunofluorescent staining of lung frozen sections
    Frozen lung tissues are prepared by inflating the lungs with optimum cutting temperature (OCT) compound through the trachea, tying off the trachea to maintain the fluid in the lung, and freezing the tissue in a disposable mold containing OCT. Frozen sections shall be cut at 5-10 µm in a cryostat microtome, mounted on Superfrost Plus microscope slides and stored in a -70 °C freezer. For detailed protocol of preparing frozen lung tissues and sections, please refer to Ling et al., 2009.
    1. Upon removal of the frozen sections from the freezer, immediately add 50 µl of 4% formaldehyde in PBS to cover the sections.
    2. Fix the tissues for 15 min at room temperature.
    3. Cover lung sections with PBS for 5 min, then drip off PBS. Repeat 3 times.
    4. Permeabilize cells by covering the sections with 0.1% Triton X-100 in PBS for 10 min (if staining for proteins that are intracellular).
    5. Rinse in PBS 3 times, 5 min each.
    6. Incubate in blocking buffer of 5% serum from secondary antibody’s host in PBS pH 7.5 for 30 min to block unspecific binding of the antibodies. Alternative blocking buffers are 1% BSA or 1% FBS in PBS.
    7. While blocking, prepare primary antibody at appropriate dilution in blocking buffer. For example, use 1:200 dilution for rabbit anti α-smooth muscle actin (α-SMA) antibody (Abcam).
    8. Incubate lung sections with diluted primary antibodies for 60 min at room temperature (or overnight at 4 °C).
    9. Rinse slides 3 times in PBS, 15 min each.
    10. Incubate specimen in fluorochrome-conjugated secondary antibodies diluted in blocking buffer for 60 min at room temperature. For example, use 1:1,000 dilution for Texas Red conjugated goat anti-rabbit IgG antibody (Abcam). From this step forward, specimen should be protected from light.
    11. Rinse slides three times in PBS, 15 min each.
    12. Coverslip slides with Prolong® Gold anti-fade reagent with or without DAPI (Figure 1). DAPI (4’,6-diamidino-2-phenylindole) is a blue-fluorescent DNA stain that serves as an ideal nuclear counterstain for fixed cells. No additional step is required for DAPI staining.


      Figure 1. Mount a coverslip on glass slides with Prolong® Gold anti-fade reagent. A. Before mounting a coverslip; B. After mounting a coverslip.

    13. Allow mounting media to cure overnight at room temperature.
    14. Observe (and photograph if necessary) an immunofluorescent stained slide by mounting it on the stage of a fluorescence microscope or a confocal microscope.

  2. H&E staining of lung sections
    Lungs are fixed and embedded in paraffin as described in Baligar et al. (2016). Lung sections shall be cut at 4-5 µm from paraffin embedded lung tissues, and mounted on glass slides.
    1. Place slides in glass staining racks. The solutions fill in square glass staining jars. For EasyDip Slide Staining System, each jar holds 80 ml reagent and 12 slides in a staining rack.
    2. Deparaffinize the sections by two successive xylene baths, 10 min each.
    3. Hydrate the lung sections by passing through decreasing concentration of alcohol baths: 2 changes of absolute alcohol, 5 min each, 95% alcohol for 2 min and 70% alcohol for 2 min.
    4. Wash briefly in distilled water.
    5. Stain in Mayer’s hematoxylin solution for 8 min.
    6. Wash in warm running tap water for 10 min.
    7. Rinse in distilled water.
    8. Counterstain in 1% eosin Y solution for 30 sec to 1 min.
    9. Dehydrate in 95% alcohol and absolute alcohol, 2 changes of 2 min each or until excess eosin is removed. Check under microscope.
    10. Clear in xylene, 2 changes of 5 min each.
    11. Coverslip slides with resinous mounting medium.
    12. Observe (and photograph if necessary) an H&E stained slide by mounting it on the stage of an optical microscope.

  3. Masson’s trichrome staining of lung sections
    1. Deparaffinize and rehydrate lung sections as above.
    2. Wash the lung sections in distilled water.
    3. Mordant in preheated Bouin Fluid for one hour at 56-60 °C or overnight at room temperature.
    4. Stain in Weigert’s iron hematoxylin solution for 10 min.
    5. Wash in running warm tap water for 10 min.
    6. Rinse in distilled water.
    7. Stain in Biebrich scarlet-acid fuchsin solution for 10-15 min.
    8. Rinse in distilled water.
    9. Differentiate in phosphomolybdic-phosphotungstic acid solution for 10 to 15 min. Observe the slides frequently with naked eyes. Once collagen-rich areas lose red color and turn to clear proceed to the next step.
    10. Transfer slides directly to aniline blue solution and stain for 5 to10 min. Rinse in distilled water and differentiate in 1% acetic acid solution for 2 to 5 min.
    11. Wash in distilled water.
    12. Dehydrate very quickly through 95% alcohol and then absolute alcohol to wipe off Biebrich scarlet-acid fuchsin staining. Clear in xylene.
    13. Mount with resinous mounting medium.
    14. Observe (and photograph if necessary) a trichrome-stained slide by mounting it on the stage of an optical microscope.

Data analysis

Lung sectioning and staining are essential methods for studying lung development or lung pathology. Immunofluorescent staining visualizes the expression and localization of interested proteins. H&E staining is most widely used in histology studies and medical diagnosis. Masson’s trichrome staining detects collagen deposition in tissues. Figure 2 shows representative images of each staining on the lung sections from mice that had been undergone bone marrow transplantation and then infected with murine gamma herpesvirus 68 for three weeks.


Figure 2. Lung section staining. The lungs were removed from C57BL/6 mice that had undergone transplantation of syngeneic EGFP-expressing bone marrow for 5 weeks, and followed by infection with murine gamma herpesvirus 68 (5 x 104 pfu/mouse) for 3 weeks. The lung had developed pneumonitis and pulmonary fibrosis as evidenced by infiltration of immune cells and deposition of collagen. A. Lung section is immunofluorescent stained with rabbit anti α-smooth muscle actin (α-SMA) antibody and α-SMA positive cells are visualized by Texas Red conjugated goat anti-rabbit IgG antibody in red. Donor bone marrow-derived cells express EGFP in green. DAPI counterstains all of the cell nuclei in blue. B. H&E staining colors cytoplasm pink and nuclei blue; C. Masson’s trichrome staining colors collagen blue, cytoplasm pink and nuclei dark brown to black.

Recipes

  1. Blocking buffer
    5% serum from secondary antibody’s host, or 1% bovine serum albumin (BSA) or fetal bovine serum (FBS) in PBS pH 7.5
  2. Eosin Y solution
    Eosin Y stock solution (1%):
    10 g eosin Y
    200 ml distilled water
    800 ml of 95% ethanol
    Mix to dissolve and store at room temperature
  3. Eosin Y working solution (0.25%)
    250 ml of eosin Y stock solution
    750 ml of 80% ethanol
    5 ml glacial acetic acid (concentrated)
    Mix well and store at room temperature
  4. Mayer’s hematoxylin solution
    50 g aluminum potassium sulfate (alum)
    1 g hematoxylin
    0.2 g sodium iodate
    1 g citric acid
    1,000 ml distilled water
    Stir to dissolve the chemicals in the order listed above
  5. Weigert’s iron hematoxylin solution
    Stock solution A:
    1 g hematoxylin
    100 ml of 95% alcohol
    Stock solution B:
    4 ml of 29% ferric chloride in distilled water
    95 ml distilled water
    1 ml hydrochloric acid, concentrated
    Weigert’s iron hematoxylin working solution:
    Mix equal parts of stock solution A and B. This working solution is stable for 3 months
  6. Phosphomolybdic-phosphotungstic acid solution
    25 ml of 5% phosphomolybdic acid
    25 ml of 5% phosphotungstic acid
  7. Aniline blue solution
    2.5 g aniline blue
    2 ml acetic acid, glacial
    100 ml distilled water

Acknowledgments

This protocol was adapted from our publication (Zhou et al., 2016). This work was supported by NIH grants AI117229, HL115618, T32HL07749 and 2UL1TR000433.

References

  1. Baligar, P., Pokhrel, S. and Mukhopadhyay, A. (2016). Experimental liver fibrosis and intrasplenic transplantation of CD45+ bone marrow cells. Bio-protocol 6(20): e1972.
  2. Ling, L. H., Rogers, S. M., Tay, V., Limmon, G. V., Dahai, Z. and Rogers, K. (2009). Comparison of various tissue-preparation techniques for cryosectioning of frozen mouse tissues. J Histotechnol 32(4): 186-189.
  3. Zhou, X., Loomis-King, H., Gurczynski, S. J., Wilke, C. A., Konopka, K. E., Ptaschinski, C., Coomes, S. M., Iwakura, Y., van Dyk, L. F., Lukacs, N. W. and Moore, B. B. (2016). Bone marrow transplantation alters lung antigen-presenting cells to promote TH17 response and the development of pneumonitis and fibrosis following gammaherpesvirus infection. Mucosal Immunol 9(3): 610-620.

简介

我们的方案描述了免疫荧光染色,苏木精和伊红染色以及Masson在肺切片上的三色染色。

背景 肺的主要功能是气体交换。肺由各种专门的细胞和组织组成,包括支气管,细支气管和肺泡,以促进气体交换。为了研究小鼠模型中的肺发育或肺部疾病,可以从小鼠中去除肺,并将其冷冻并嵌入最佳切割温度(OCT)化合物或化学保藏并包埋在石蜡中。为了保持肺组织结构,我们用1ml OCT化合物填充肺,用于制备冷冻切片,或用于石蜡切片的10%缓冲福尔马林(Zhou等人,2016)。然后将肺切片从冷冻或石蜡包埋的肺切片并安装到载玻片上用于制备染色。我们使用冷冻组织切片进行基于抗体的免疫荧光染色,并使用石蜡包埋切片进行苏木精和伊红(H&E)染色或Masson三色染色。冷冻切片更快地准备免疫荧光染色,大多数抗体在冷冻切片上工作良好。石蜡切片也可用于免疫荧光染色,但需要脱蜡,补液和抗原检索。即使在抗原检索后,一些抗体在石蜡切片上也不能很好地工作。然而,石蜡样品可以在室温下储存很长时间,并且可以容易地切成非常薄的部分。石蜡切片保留比冷冻切片更好的组织形态,因此它们对于H&E或三色染色更好。
 免疫荧光染色是一种使用荧光团观察特异性结合其靶蛋白的抗体位置的免疫组织化学。 H&E染色是组织学和医学诊断中使用最广泛的染色。这种染色方法涉及将细胞核蓝色和细胞质粉红色变成红色的血红素和伊红Y。 Masson的三色染色是用于检测组织中胶原纤维的三色染色。染色产生蓝色胶原蛋白,深棕色至黑色细胞核和红色背景。

关键字:免疫荧光染色, 苏木精和伊红染色, Masson三色染色, 冷冻组织切片, 石蜡包埋组织切片, 抗体

材料和试剂

  1. Fisherbrand Superfrost Plus显微镜载玻片(Fisher Scientific,目录号:12-550-15)
  2. 一次性模具
  3. 盖玻片
  4. C57BL/6小鼠
  5. Tissue-Tek OCT化合物(SAKURA FINETEK,目录号:4583)
  6. 甲醛,37-40%ACS(新来者供应,目录号:1089)
  7. Triton X-100(Sigma-Aldrich,目录号:X100)
  8. 磷酸盐缓冲盐水(PBS,pH 7.4)(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
  9. 来自二抗的宿主的血清
  10. 一抗:兔抗α-平滑肌肌动蛋白(α-SMA)抗体(Abcam,目录号:ab5694)
  11. 二次试剂:德克萨斯红色缀合山羊抗兔IgG抗体(Abcam,目录号:ab6719)
  12. Prolong ®金抗褪色试剂(Cell Signaling Technology,目录号:9071)或用DAPI(Cell Signaling Technology,目录号:8961)
  13. 石蜡
  14. 乙醇(Newcomer Supply,目录号:10841)
  15. 二甲苯(Newcomer Supply,目录号:1446)
  16. S安装介质(Newcomer Supply,目录号:6750)
  17. Bouin Fluid(Newcomer Supply,目录号:1020)
  18. Biebrich猩红酸品红溶液(Sigma-Aldrich,目录号:HT151)
  19. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A3912)
  20. 胎牛血清(FBS)(GE Healthcare,Hyclone TM,目录号:SH30396.03)
  21. 曙红Y(Sigma-Aldrich,目录号:E4009)
  22. 乙酸,冰川(Sigma-Aldrich,目录号:A6283)
  23. 硫酸铝钾(Sigma-Aldrich,目录号:237086)
  24. 苏木精(Sigma-Aldrich,目录号:H3136)
  25. 碘酸钠(Sigma-Aldrich,目录号:S4007)
  26. 柠檬酸(Sigma-Aldrich,目录号:251275)
  27. 氯化铁(Sigma-Aldrich,目录号:157740)
  28. 盐酸(Sigma-Aldrich,目录号:320331)
  29. 磷钼酸溶液(Sigma-Aldrich,目录号:HT153)
  30. 磷钨酸溶液(Sigma-Aldrich,目录号:HT152)
  31. 苯胺蓝(Sigma-Aldrich,目录号:415049)
  32. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  33. 磷酸二氢钠(NaH 2 PO 4)(Sigma-Aldrich,目录号:S8282)
  34. 阻塞缓冲区(见配方)
  35. 曙红Y溶液(见配方)
  36. 曙红Y工作液(0.25%)(见配方)
  37. Mayer的苏木精溶液(参见食谱)
  38. Weigert的铁苏木精溶液(参见食谱)
  39. 磷钼铁磷酸溶液(见配方)
  40. 苯胺蓝溶液(参见食谱)

设备

  1. 冷冻切片机
  2. -70°C冷冻箱
  3. 荧光显微镜或共聚焦显微镜
  4. 染色罐和滑架,例如EasyDip幻灯片染色系统(Newcomer Supply,目录号:5300KIT)
  5. 光学显微镜

程序

  1. 肺冷冻切片的免疫荧光染色 冷冻的肺组织是通过气管充气具有最佳切割温度(OCT)化合物的肺来制备的,将气管夹紧以保持肺中的流体,并在含有OCT的一次性模具中冷冻组织。冷冻切片应在低温恒温切片机中切成5-10微米,安装在Superfrost Plus显微镜载玻片上并储存在-70℃的冷冻箱中。关于制备冷冻肺组织和切片的详细方案,请参阅Ling等人,2009年。
    1. 从冰箱中取出冷冻切片后,立即加入50μl4%甲醛的PBS溶液以覆盖各部分
    2. 在室温下将组织固定15分钟。
    3. 用PBS封闭肺切片5分钟,然后滴入PBS。重复3次。
    4. 通过在PBS中用0.1%Triton X-100覆盖切片10分钟(如果细胞内蛋白质染色),可以渗透细胞。
    5. 在PBS中冲洗3次,每次5分钟。
    6. 在PBS pH 7.5中从二次抗体宿主的5%血清的封闭缓冲液中孵育30分钟,以阻断抗体的非特异性结合。替代阻断缓冲液是PBS中的1%BSA或1%FBS。
    7. 当阻断时,在封闭缓冲液中适当稀释制备一抗。例如,使用1:200稀释用于兔抗α-平滑肌肌动蛋白(α-SMA)抗体(Abcam)。
    8. 在室温下(或4℃过夜),用稀释的一抗孵育肺切片60分钟。
    9. 在PBS中漂洗3次,每次15分钟。
    10. 在室温下用封闭缓冲液稀释60分钟的荧光染料缀合的二次抗体孵育样品。例如,对于德克萨斯红缀合的山羊抗兔IgG抗体(Abcam)使用1:1,000稀释。从这一步向前,标本应避光
    11. 在PBS中漂洗三次,每次15分钟。
    12. 带有或不带DAPI的Prolong®镀金抗褪色试剂(图1)。 DAPI(4',6-二脒基-2-苯基吲哚)是一种蓝色荧光DNA染色剂,可作为固定细胞的理想核复染料。 DAPI染色不需要额外的步骤。


      图1.使用Prolong ®金防褪色试剂在玻片上安装盖玻片。 A.安装盖玻片之前。 B.安装盖玻片后。

    13. 允许介质在室温下固化过夜。
    14. 通过将免疫荧光染色载玻片安装在荧光显微镜或共聚焦显微镜的平台上,观察(如有必要)进行照相(并进行照相)。

  2. 肺切片的H& E染色 如Baligar等人所述,将肺固定并包埋在石蜡中。 (2016)。肺切片应从石蜡包埋的肺组织切成4-5μm,并安装在载玻片上。
    1. 将玻片放在玻璃染色架上。溶液装满方形玻璃染色瓶。对于EasyDip幻灯片染色系统,每个容器在染色架上容纳80ml试剂和12个载玻片。
    2. 通过两个连续的二甲苯浴将馏分脱蜡,每次10分钟。
    3. 通过不断浓缩的酒精水浴使肺部水分流失:2次绝对酒精,5分钟,95%酒精2分钟,70%酒精2分钟。
    4. 在蒸馏水中短暂洗涤。
    5. 在Mayer的苏木精溶液中染色8分钟
    6. 在温暖的自来水中洗涤10分钟。
    7. 用蒸馏水冲洗。
    8. 在1%曙红Y溶液中复染30秒至1分钟
    9. 在95%酒精和无水酒精中脱水,2次每次2分钟或直到除去过量的曙红。在显微镜下检查
    10. 二甲苯清除2次,每次5分钟。
    11. 带树脂安装介质的盖板滑块。
    12. 通过将H& E染色的载玻片安装在光学显微镜的平台上,观察(如有必要)进行照相(如有需要)。

  3. Masson的三段染色肺切片
    1. 如上所述对肺部进行脱蜡和再水合。
    2. 用蒸馏水清洗肺部。
    3. 媒染剂在预热的Bouin流体中在56-60℃下持续1小时,或在室温下过夜
    4. 在Weigert的铁苏木精溶液中染色10分钟
    5. 在运行温水自来水中洗涤10分钟。
    6. 用蒸馏水冲洗。
    7. 在Biebrich猩红酸品红色溶液中染色10-15分钟。
    8. 用蒸馏水冲洗。
    9. 在磷钼磷钨酸溶液中分解10至15分钟。用肉眼观察幻灯片。一旦胶原蛋白丰富的区域失去红色,转而清除,进入下一步。
    10. 将载玻片直接转移至苯胺蓝溶液并染色5至10分钟。用蒸馏水冲洗,并在1%乙酸溶液中分化2〜5分钟
    11. 用蒸馏水冲洗。
    12. 通过95%酒精脱水,然后用绝对酒精擦去Biebrich猩红酸品红染色。二甲苯清除。
    13. 搭载树脂固定介质。
    14. 通过将三色染色的载玻片安装在光学显微镜的平台上,观察(如有必要)(如有必要)拍摄。

数据分析

肺切片和染色是研究肺发育或肺部病理学的重要手段。免疫荧光染色显示感兴趣的蛋白质的表达和定位。 H& E染色最广泛地用于组织学研究和医学诊断。 Masson的三色染色检测组织中的胶原沉积。图2显示了已经经历骨髓移植的小鼠的肺切片上的每个染色的代表性图像,然后用鼠疱疹病毒68感染三周。


图2.肺部染色从经历了同基因EGFP表达骨髓移植5周的C57BL/6小鼠中取出肺,然后感染鼠疱疹病毒68(5× 10小时/小时)3周。由于免疫细胞的浸润和胶原蛋白的沉积,肺部发生了肺炎和肺纤维化。 A.肺部是用兔抗α-平滑肌肌动蛋白(α-SMA)抗体进行免疫荧光染色,α-SMA阳性细胞通过德克萨斯红缀合的山羊抗兔IgG抗体以红色显现。供体骨髓来源的细胞以绿色表达EGFP。 DAPI将所有细胞核重新染色为蓝色。 B.H& E染色染色细胞质粉红色和核蓝色; C. Masson的三色染色将胶原蛋白蓝色,细胞质粉红色和细胞核深棕色至黑色。

食谱

  1. 阻塞缓冲区
    来自第二抗体宿主的5%血清或PBS pH7.5中的1%牛血清白蛋白(BSA)或胎牛血清(FBS)
  2. Eosin Y解决方案
    Eosin Y库存解决方案(1%):
    10 g伊红Y
    200毫升蒸馏水
    800 ml的95%乙醇 混合溶解并在室温下储存
  3. 曙光Y工作液(0.25%)
    250毫升伊红Y储备溶液
    750毫升80%乙醇 5 ml冰醋酸(浓缩)
    混合好并在室温下储存
  4. Mayer的苏木精溶液
    50克硫酸铝钾(明矾)
    1克苏木精
    0.2克碘酸钠
    1克柠檬酸
    1000毫升蒸馏水
    搅拌以上述顺序溶解化学品
  5. Weigert的铁苏木精溶液
    库存解决方案A:
    1克苏木精
    100毫升95%酒精
    库存解决方案B:
    4毫升29%的氯化铁在蒸馏水中 95 ml蒸馏水
    1ml盐酸,浓缩, Weigert的铁苏木精工作液:
    混合相当部分的储备溶液A和B。该工作溶液稳定3个月
  6. 磷钼铁磷酸溶液
    25毫升5%磷钼酸
    25毫升5%磷钨酸
  7. 苯胺蓝溶液
    2.5 g苯胺蓝色
    2毫升醋酸,冰川
    100ml蒸馏水

致谢

该协议是从我们的出版物(Zhou等人,2016)改编而成。这项工作得到NIH授权AI117229,HL115618,T32HL07749和2UL1TR000433的支持。

参考

  1. Baligar,P.,Pokhrel,S。和Mukhopadhyay,A.(2016)。  CD45 + 骨髓细胞的实验性肝纤维化和脾内移植。 6(20):e1972。 >
  2. Ling,LH,Rogers,SM,Tay,V.,Limmon,GV,Dahai,Z.and Rogers,K。(2009)。  用于冷冻小鼠组织冷冻切片的各种组织制备技术的比较 His Histechnol 32 (4):186-189。
  3. Zhou,X.,Loomis-King,H.,Gurczynski,SJ,Wilke,CA,Konopka,KE,Ptaschinski,C.,Coomes,SM,Iwakura,Y.,van Dyk,LF,Lukacs,NW和Moore,BB (2016)。骨髓移植改变肺抗原呈递细胞促进TH17反应,以及伽pes病毒感染后肺炎和纤维化的发展。粘膜免疫学9(3):610-620。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhou, X. and Moore, B. B. (2017). Lung Section Staining and Microscopy. Bio-protocol 7(10): e2286. DOI: 10.21769/BioProtoc.2286.
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