Ear Inflammation and Whole-mount Ear Staining

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The recruitment of circulating neutrophils from the bloodstream to the site of inflammation represents one of the earliest events during an innate immune response. During this response, neutrophils tether and roll along the vessel walls before transmigrating across the endothelium into the interstitial space to exert their functions. Here, we describe a protocol for the staining of intravascular and tissue-localized neutrophils following contact sensitization of the skin with croton oil. Visualization of the neutrophilic distribution in skin provides for a better interpretation of the local immune response.

Keywords: Neutrophils(中性白细胞), Ear inflammation(耳朵发炎), Skin inflammation(皮肤炎症)


Characterisation of neutrophil distribution within the skin following inflammation represents an important avenue for the understanding of their specialized functions. Even though the recruitment of neutrophils to the inflamed skin has been widely characterized by flow cytometry (Hampton et al., 2015; Stock et al., 2014), such technique provides limited insight on the intravascular versus interstitial localization of neutrophils. Recirculating and tissue-localized neutrophils exhibit different phenotypes and functions, which necessitates their discrimination to identify key players of the local immune response. With the development of intravital microscopy (IVM), the direct visualization of fluorescently tagged immune cells in vivo is made possible. However, due to the high cost of IVM, the accessibility of this powerful imaging tool to researchers is limited. Here, we present an alternative protocol for the high resolution static imaging of neutrophils in skin by making use of cost-effective reagents and commonly available confocal laser-scanning microscopy.

Materials and Reagents

  1. Cotton swabs
  2. SterilinTM Petri dish (90 mm) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 101IRR )
  3. 24-well cell culture plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 142475 )
  4. Glass slides (Biomedical Sciences Institutes, catalog number: BMH.880103 )
  5. FisherbrandTM cover glass (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-545-81 )
  6. Clear nail polish
  7. Mice
  8. Croton oil (Sigma-Aldrich, catalog number: C6719 )
  9. Veet hair removal cream
  10. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  11. Acetone (Thermo Fisher Scientific, Fisher Scientific, catalog number: 67-64-1 )
  12. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: 10735108001 )
  13. Sodium azide (Sigma-Aldrich, catalog number: S2002 )
  14. Antibodies
    1. PE anti-mouse Ly-6G antibody (clone 1A8) (BioLegend, catalog number: 127607 )
    2. PE rat IgG2a, κ Isotype control antibody (clone RTK2758) (BioLegend, catalog number: 400507 )
    3. APC anti-mouse CD31 (clone MEC 13.3) (BD, PharmingenTM, catalog number: 561814 )
  15. Mounting medium (ibidi, catalog number: 50001 )
  16. DAPI (Sigma-Aldrich, catalog number: D9564 )
  17. Stain and wash buffer (1% BSA in PBS) (see Recipes)


  1. Pipette (Eppendorf, model: Eppendorf Research plus )
  2. Dissection scissors (Roboz Surgical Instrument, catalog number: RS-6702 )
  3. Forceps (Roboz Surgical Instrument, catalog number: RS-5240 ; RS-5135 )
  4. Shaker (Labnet)
  5. Zeiss LSM 510 Meta confocal microscope (Zeiss)


  1. ImageJ software (National Institutes of Health) (https://imagej.nih.gov/ij/index.html)


  1. Apply 20 μl of freshly prepared 1% croton oil (in acetone) on the dorsal side of each ear with a pipette.
  2. Sacrifice mice after 3 h. Remove hair on dorsal side of ear with Veet using a cotton swab (Figure 1A). Wash away Veet with PBS using a cotton swab after 3 min. Repeat until Veet is completely removed (Figure 1B).

    Figure 1. Removal of hair from mouse ear. A. Application of Veet on the dorsal side of ear using a cotton swab. B. After 3 min, Veet is completely removed with PBS using a cotton swab.

  3. Harvest ear. Split ears into halves on a Petri dish using forceps, and collect the dorsal side (Figure 2).

    Figure 2. Harvesting of ear sample. A. Harvest ear with scissors and forceps. B. Ear is split into halves using forceps.

  4. Transfer the dorsal ear halves into a 24-well plate and fix with 1 ml acetone at -20 °C for 20 min.
  5. Remove the acetone and wash 3 times with 1 ml of stain and wash buffer in the 24-well plate at room temperature.
  6. Stain the ear halves in a 24-well plate by adding 50 μl of Ly6G PE/Isotype control PE and CD31 APC (1:100 in stain and wash buffer) directly onto the ear halves. Incubate for 1 h at room temperature with shaking.
  7. Wash 3 times with 1 ml of stain and wash buffer in the 24-well plate at room temperature.
  8. Transfer ear halves onto cover glass. Mount with 10 μl mounting medium containing DAPI (1:1,000) onto glass slides and seal with clear nail polish (Figure 3).

    Figure 3. Mounting of ear sample onto glass slide. A. Mounting medium is added onto ear halves. B. Ear halves on cover glasses are mounted onto glass slide.

  9. Visualize under confocal microscope. Image under 20x objective, with the HeNe 543 (Cy3) and HeNe633 (Cy5) laser (Figure 4).

    Figure 4. Immunofluorescence staining of neutrophils in whole-mount ear 3 h after topical application of croton oil. Neutrophils were visualized with anti-Ly6G (red), and blood vessels with anti-CD31 (green). Scale bar, 50 μm.

Data analysis

  1. Download ImageJ software from https://imagej.nih.gov/ij/index.html.
  2. Open image in ImageJ.
  3. Adjust brightness or contrast of image if necessary:
    Image → Adjust → Brightness/Contrast
  4. To overlay the two fluorescence channels:
    Image → Stacks → Stack to Images
    Image → Color → Merge channels…
  5. Export image as TIFF file.


  1. Hair removal is important to minimize autofluorescence during imaging.
  2. Isotype control ensures specificity of Ly6G staining.


  1. Stain and wash buffer (1% BSA in PBS)
    1 g BSA
    200 μl 10% sodium azide
    100 ml PBS


This protocol was adapted from the experimental procedures of previously published paper in Gunawan et al. (2015). This work was supported by the National Research Council of Singapore (NMRC/CBRG/0057/2014).


  1. Gunawan, M., Venkatesan, N., Loh, J. T., Wong, J. F., Berger, H., Neo, W. H., Li, L. Y., La Win, M. K., Yau, Y. H., Guo, T., See, P. C., Yamazaki, S., Chin, K. C., Gingras, A. R., Shochat, S. G., Ng, L. G., Sze, S. K., Ginhoux, F. and Su, I. H. (2015). The methyltransferase Ezh2 controls cell adhesion and migration through direct methylation of the extranuclear regulatory protein talin. Nat Immunol 16(5): 505-516.
  2. Hampton, H. R., Bailey, J., Tomura, M., Brink, R. and Chtanova, T. (2015). Microbe-dependent lymphatic migration of neutrophils modulates lymphocyte proliferation in lymph nodes. Nat Commun 6: 7139.
  3. Stock, A. T., Smith, J. M. and Carbone, F. R. (2014). Type I IFN suppresses Cxcr2 driven neutrophil recruitment into the sensory ganglia during viral infection. J Exp Med 211(5): 751-759.



[背景] 炎症后中性粒细胞分布在皮肤中的表征代表了理解其专门功能的重要途径。即使通过流式细胞术广泛表征嗜中性粒细胞在炎症皮肤中的募集(Hampton等人,2015; Stock em 。,2014),这种技术提供有限的洞察中性粒细胞的血管内与间质定位。再循环和组织定位中性粒细胞展示不同的表型和功能,这使得他们的歧视需要识别当地免疫反应的关键参与者。随着活体显微镜(IVM)的发展,使得荧光标记的免疫细胞在体内的直接可视化成为可能。然而,由于IVM的高成本,这种强大的成像工具对研究人员的可访问性是有限的。在这里,我们提出了高分辨率静电成像的中性粒细胞在皮肤中使用成本效益试剂和通常可用的共聚焦激光扫描显微镜的替代协议。

关键字:中性白细胞, 耳朵发炎, 皮肤炎症


  1. 棉签
  2. 培养皿(90mm)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:101IRR)
  3. 24孔细胞培养板(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:142475)
  4. 玻璃载玻片(Biomedical Sciences Institutes,目录号:BMH.880103)
  5. Fisherbrand TM盖玻片(Thermo Fisher Scientific,Fisher Scientific,目录号:12-545-81)
  6. 清除指甲油
  7. 小鼠
  8. 巴豆油(Sigma-Aldrich,目录号:C6719)
  9. Veet脱毛膏
  10. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10010023)
  11. 丙酮(Thermo Fisher Scientific,Fisher Scientific,目录号:67-64-1)
  12. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:10735108001)
  13. 叠氮化钠(Sigma-Aldrich,目录号:S2002)
  14. 抗体
    1. PE抗小鼠Ly-6G抗体(克隆1A8)(BioLegend,目录号:127607)
    2. PE大鼠IgG2a,κ同种型对照抗体(克隆RTK2758)(BioLegend,目录号:400507)
    3. APC抗小鼠CD31(克隆MEC 13.3)(BD,Pharmingen TM ,目录号:561814)
  15. 安装介质(ibidi,目录号:50001)
  16. DAPI(Sigma-Aldrich,目录号:D9564)
  17. 染色和洗涤缓冲液(1%BSA的PBS溶液)(参见配方)


  1. 移液管(Eppendorf,型号:Eppendorf Research plus)
  2. 解剖剪刀(Roboz Surgical Instrument,目录号:RS-6702)
  3. 钳(Roboz Surgical Instrument,目录号:RS-5240; RS-5135)
  4. 振动器(Labnet)
  5. Zeiss LSM 510 Meta共焦显微镜(Zeiss)


  1. ImageJ软件(美国国家卫生研究院)( https://imagej.nih .gov/ij/index.html


  1. 用移液管在每只耳朵的背侧涂20μl新鲜制备的1%巴豆油(丙酮溶液)。
  2. 3小时后牺牲小鼠。使用棉签用Veet去除耳朵背侧的毛发(图1A)。 3分钟后用棉签用PBS洗去Veet。重复,直到Veet完全去除(图1B)。

    图1.从小鼠耳朵取出头发。 A.使用棉签在耳背侧应用Veet。 B. 3分钟后,用棉签用PBS完全除去Veet
  3. 收获耳朵。使用镊子在培养皿上将耳朵分成两半,收集背侧(图2)。

    图2.收集耳朵样本。A.用剪刀和镊子收获耳朵。 B.使用镊子将耳朵分成两半。

  4. 将背侧耳朵转移到24孔板中,并用1ml丙酮在-20°C固定20分钟。
  5. 取出丙酮,在室温下用24孔板中的1ml染色剂和洗涤缓冲液洗涤3次
  6. 通过添加50微升Ly6G PE /同种型对照PE和CD31 APC(染色和洗涤缓冲液中的1:100)到24孔板上的耳朵半部分染色。在室温下振荡孵育1小时
  7. 在室温下在24孔板中用1ml染色剂和洗涤缓冲液洗涤3次
  8. 将半耳朵转移到玻璃罩上。用10μl含DAPI(1:1,000)的固定介质安装到载玻片上,并用透明指甲油密封(图3)。

    图3.将耳朵样品安装到载玻片上。 A.将安装介质添加到耳朵半部。 B.玻璃上的耳朵半部分安装在玻璃载玻片上
  9. 在共聚焦显微镜下可视化。使用HeNe 543(Cy3)和HeNe633(Cy5)激光器(图4)在20x物镜下的图像。



  1. https://imagej.nih.gov/ij/下载ImageJ软件index.html
  2. 在ImageJ中打开图片。
  3. 如有必要,调整图像的亮度或对比度:
  4. 覆盖两个荧光通道:
  5. 将图片导出为TIFF文件。


  1. 毛发去除对于最小化成像期间的自发荧光是重要的
  2. 同种型对照确保Ly6G染色的特异性


  1. 染色和洗涤缓冲液(1%BSA的PBS溶液) 1 g BSA
    100 ml PBS


该协议改编自Gunawan等人以前发表的论文的实验程序。 (2015)。这项工作得到了新加坡国家研究委员会的支持(NMRC/CBRG/0057/2014)。


  1. Gunawan,M.,Venkatesan,N.,Loh,JT,Wong,JF,Berger,H.,Neo,WH,Li,LY,La Win,MK,Yau,YH,Guo, ,S,Chin,KC,Gingras,AR,Shochat,SG,Ng,LG,Sze,SK,Ginhoux,F.and Su,IH(2015)。  甲基转移酶Ezh2通过核外调节蛋白talin的直接甲基化来控制细胞粘附和迁移。 Immunol 16(5):505-516。
  2. Hampton,HR,Bailey,J.,Tomura,M.,Brink,R.和Chtanova,T。(2015)。  嗜中性粒细胞的微生物依赖性淋巴迁移调节淋巴结中的淋巴细胞增殖。 Nat Commun 6:7139.
  3. 股票,AT,史密斯,JM和Carbone,FR(2014)。  I型IFN在病毒感染期间抑制Cxcr2驱动的嗜中性粒细胞募集到感觉神经节中。 211(5):751-759。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Loh, J. T., Gunawan, M. and Su, I. (2016). Ear Inflammation and Whole-mount Ear Staining. Bio-protocol 6(20): e1967. DOI: 10.21769/BioProtoc.1967.

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