Quantification of Rae-1 Staining Intensity in Glomeruli

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Journal of Leukocyte Biology
Mar 2015


NKG2D is expressed on all NK cells and on subsets of NKT, CD8, CD4 and γδ T cells. NKG2D is activated by NKG2D ligands, a stress-induced family of MHC-I-like proteins. These ligands are upregulated on stressed/infected cells and are not widely expressed on healthy adult tissue. NKG2D ligands have been widely studied as potential targets for immunotherapeutic approaches in cancer and auto-immune diseases such as Systemic Lupus Erythematosus. Here we describe a method for quantifying the expression levels of the Rae-1 NKG2D ligand in the glomeruli of healthy and diseased individuals via a novel algorithm. The MRL mouse was used as a positive control strain as it spontaneously generates a lupus-like phenotype, one of the main effects of which is severe glomerulonephritis. MRL/MpJ mice develop this phenotype spon¬taneously at ~12 months of age, whereas MRL/MpJlpr mice, which have the same genetic background but which generated a spontaneous homozygous mutation in the Fas allele, develop similar but more severe symptoms by ~3-4 months of age (Spada et al., 2015).

Keywords: NKG2D ligands (NKG2D配体), Lupus (系统性红斑狼疮), Glomerulonephritis (肾小球肾炎), MRL/MpJlpr (MRL / mpjlpr)

Materials and Reagents

  1. Standard Cryomold (Electron Microscopy Sciences, Tissue-Tek, catalog number: 4557 )
  2. Female MRL/MpJ [3 months (healthy) and 12 months (diseased)] and MRL/MpJ-Faslpr/J [9 weeks (pre-disease) and 3 months (diseased)]
  3. Peroxidase Block (Dako, catalog number: K4009 )
  4. Goat anti-mouse Rae-1γ (R&D Systems, catalog number: AF1136 )
  5. Goat Histofine Simple Stain kit (NICHIREI CORPORATION, catalog number: 414161F )
  6. AEC + Substrate-Chromogen (Dako, catalog number: K3464 )
  7. PBS (Sigma-Aldrich, catalog number: P5493 )
  8. Aqueous mounting medium (Vector Labs, catalog number: H-5501 )
  9. Acetone (Sigma-Aldrich, catalog number: 320110 )
  10. Tissue-Freezing Medium (Leica Biosystems Nussloch GmbH, catalog number: 14020108926 )
  11. Haematoxylin (Sigma-Aldrich, catalog number: H9627 )


  1. Leica Leitz DM RB Microscope (Leica Microsystems) with an adapted Olympus camera (Olympus Imaging America, model: DP70)
  2. Macintosh or Windows PC


  1. Photoshop CS6 (Adobe)
  2. Matlab v. 2013a (MathWorks)


  1. Sample preparation
    1. Kidneys were removed via a ventral midline incision into the abdomen of a sacrificed mouse. The intestines were retracted laterally to expose the animal’s kidneys. The kidneys are freed from the surrounding tissue and excised.
    2. Kidneys were briefly washed in 1x PBS, placed within a cryo-mold, covered with OCT tissue-freezing medium and snap frozen by placing the cryo-mold on top of liquid nitrogen.
    3. The frozen sections kidneys were then cut in 10 μm sections using a cryostat.
      1. For general guidelines on how to cut sections please refer to the work performed by Fischer et al., (2008).
    4. Samples to be processed were thawed at RT for 10 min and then fixed with ice-cold 100% acetone for 10 min at -20 degrees.


    1. Isotype-incubated slides were used as negative controls.
    2. All wash steps were performed in coplin jars.
    1. Sections were washed for 2 x 5 min in 1x PBS.
    2. Endogenous peroxidase was blocked using DAKO’s endogenous peroxidase blocker for 7 min at RT as per manufacturer’s instructions.
    3. Sections were washed for 2 x 5 min in 1x PBS.
    4. Sections were incubated with Goat anti-mouse Rae-1γ antibody (1/100 dilution in 1x PBS with 10% FBS and 2.5% BSA) overnight at 4 °C.
    5. Sections were washed for 2 x 5 min in 1x PBS.
    6. Sections were incubated with Goat Histofine Simple Stain kit for 30 min at RT as per manufacturer’s instructions.
    7. Sections were washed for 2 x 5 min in 1x PBS.
    8. Sections were stained with AEC+ Substrate-Chromogen for 15-20 min at RT as per manufacturer’s instructions.
    9. Reaction was stopped by rinsing the slides gently with distilled water from a wash bottle (do not focus flow directly on tissue).
    10. Sections were hematoxylin counterstained for 3 min.
    11. Reaction was stopped by placing slides under running tap water for 10 min.
    12. 1 drop of aqueous mounting medium was placed on each slide and coverslip was carefully added as not to form bubbles.
    13. Slides were allowed to dry for 1 h at RT.
    14. Chromogen deposition was measured by quantitative immunohistochemistry by use of an established method (see section B "Image analysis").
    15. Images of glomeruli (100x magnification) were acquired under a Leica microscope (vertical Leitz DM RB) with an adapted Olympus (DP70) camera; image files were saved in a tagged image file format (antibody stained images are referred to as EXP and control images are referred to as CONTROL).

  2. Image analysis
    All images were then opened in Photoshop and processed as following [as per Matkowskyj et al., (2000)]:
    1. Open Image in Photoshop.
    2. Define size parameters for Marquee tool.
      1. Double click Marquee icon.
      2. Define area in pixels (200 x 200 pixel region).
      Note: The area has to be chosen arbitrarily as the smallest possible area in which an entire glomerulus can be visualized. The user must then proceed in selecting only those glomeruli which fit comfortably in this area (see step B7). The same pixel area must be used for all samples in each group. In our case, the 200 x 200 pixel region proved to be the best-fit.
    3. Identify region to be analyzed with Marquee tool.
    4. Store in a new file.
      1. Function “Edit” select “Copy”.
      2. Function “Edit” select “New File”.
      3. In the new file select “Paste”.
      4. Function “Save” select tiff for file type.
    5. Store a new file in Matlab folder.
    6. Open Matlab.
      At the cursor (») type
      »a= double(imread(‘EXP’,‘tiff’));
      »b=double(imread(‘CONTROL', ‘tiff’));
      »ea=norm(a(:,:,1)) +norm(a(:,:,2)) +norm(a(:,:,3));
      »eb=norm(b(:,:,1)) +norm(b(:,:,2)) +norm(b(:,:,3));
    7. Following this protocol the amount of chromogen/pixel was determined by selecting glomeruli (25 glomeruli/group) in a 200 x 200 pixel region and subtracting the mathematical energy (EM - calculated at step B6) of the control slide (CONTROL - not exposed to primary antibody) from that of a homologous glomerulus on the experimental slide (EXP - exposed to Rae-1 antibody).
      Note: The 200 x 200 pixel region was used because it proved to be the region size which was capable of including the glomeruli of all samples in the most homologous way possible. This size must be adapted according to the samples at hand.

Representative data

Figure 1. Example of IHC Rae-1 staining (red) in kidneys from 1 year old MRL/MpJ mouse model taken at 10x. A. Negative control slide; B. Slide incubated with Rae-1 antibody.

Figure 2. Example 200 x 200 pixel regions extracted from glomeruli at 100x magnification. A. Glomerulus from negative control slide; B. Glomerulus from Rae-1 incubated slide.

Representative results are from the quantification of Rae-1 in the glomeruli of healthy, pre-disease and diseased models of MRL mice. Using this technique, we were capable of determining that Rae-1 expression levels were higher in diseased models of MRL mice compared to healthy and pre-diseased ones (Spada et al., 2015).

Figure 3. Quantification of Rae-1γ staining in MRL mouse glomeruli. Data were analyzed with a 2-tailed, unpaired Student’s t-test (mean 6 SD; n = 5/group; **P, 0.0001; *P, 0.0025).


  1. This technique allows for the quantification of the absolute amount of Rae-1 antibody chromogen per pixel in the glomeruli of healthy and diseased mice. However this technique is not limited to the glomeruli nor to the Rae-1 antibody, as it can be modified as needed for the determination of staining intensity of any antibody in any cellular region or structure.
  2. It is crucial that all samples (including negative controls) be processed and analyzed simultaneously in order to minimize variation in the staining results.


This work was supported, in part, by grants from the Spanish Ministry of Economy and Competitiveness (SAF-2008-00471 and SAF-2011-23639; to D. F. B.), the Madrid regional government (CCG08-CSIC/SAL-3451; to D. F. B.), and the Spanish Ministry for Health, Social Services and Equality, Carlos III Health Institute Cooperative Research Thematic Network program: Research Network in Inflammation and Rheumatic Diseases (RIER; RD08/ 0075/0015; to D. F. B.) and Spanish National Biobank Network (RD09/0076/00101 to the FJD). R. S. was supported by an FPI predoctoral fellowship from the Spanish Ministry of Economy and Competitiveness. We want to thank the work from Matkowskyj et al. (2000) which served as the basis for this protocol.


  1. Fischer, A. H., Jacobson, K. A., Rose, J. and Zeller, R. (2008). Cryosectioning tissues. CSH Protoc 2008: pdb prot4991.
  2. Matkowskyj, K. A., Schonfeld, D. and Benya, R. V. (2000). Quantitative immunohistochemistry by measuring cumulative signal strength using commercially available software photoshop and matlab. J Histochem Cytochem 48(2): 303-312.
  3. Spada, R., Rojas, J. M., Perez-Yague, S., Mulens, V., Cannata-Ortiz, P., Bragado, R. and Barber, D. F. (2015). NKG2D ligand overexpression in lupus nephritis correlates with increased NK cell activity and differentiation in kidneys but not in the periphery. J Leukoc Biol 97(3): 583-598.


NKG2D在所有NK细胞上和在NKT,CD8,CD4和γδT细胞的子集上表达。 NKG2D被NKG2D配体激活,NKG2D配体是应激诱导的MHC-I样蛋白家族。这些配体在应激/感染的细胞上上调,并且不在健康成人组织上广泛表达。 NKG2D配体已被广泛研究作为在癌症和自身免疫疾病如系统性红斑狼疮中的免疫治疗方法的潜在靶标。在这里我们介绍一种方法通过一个新的算法,量化健康和患病个体的肾小球Rae-1 NKG2D配体的表达水平。 MRL小鼠用作阳性对照菌株,因为其自发产生狼疮样表型,其中一个主要影响是严重的肾小球肾炎。 MRL/MpJ小鼠在约12个月龄时自发产生该表型,而具有相同遗传背景但在Fas等位基因中产生自发纯合突变的MRL/MpJlpr小鼠发展类似但更严重的症状〜3 -4个月龄(Spada等人,2015)。

关键字:NKG2D配体, 系统性红斑狼疮, 肾小球肾炎, MRL / mpjlpr


  1. Standard Cryomold(Electron Microscopy Sciences,Tissue-Tek,目录号:4557)
  2. 女性MRL/MpJ [3个月(健康)和12个月(患病)]和MRL/MpJ-Fas lpr/J [9周(疾病前)和3个月/>
  3. 过氧化物酶块(Dako,目录号:K4009)
  4. 山羊抗小鼠Rae-1γ(R& D Systems,目录号:AF1136)
  5. 山羊Histofine简单染色试剂盒(NICHIREI CORPORATION,目录号:414161F)
  6. AEC +底物 - 色原(Dako,目录号:K3464)
  7. PBS(Sigma-Aldrich,目录号:P5493)
  8. 水性封固剂(Vecor Labs,目录号:H-5501)
  9. 丙酮(Sigma-Aldrich,目录号:320110)
  10. 组织冷冻培养基(Leica Biosystems Nussloch GmbH,目录号:14020108926)
  11. 苏木精(Sigma-Aldrich,目录号:H9627)


  1. Leica Leitz DM RB显微镜(Leica Microsystems),带有适配的奥林巴斯相机(Olympus Imaging America,型号:DP70)
  2. Macintosh或Windows PC


  1. Photoshop CS6(Adobe)
  2. Matlab v。2013a(MathWorks)


  1. 样品准备
    1. 通过腹侧中线切口将肾脏移除到腹部 的牺牲小鼠。肠横向缩回以暴露 ?动物的肾脏。肾脏从周围组织释放 ?并切除。
    2. 将肾脏在1x PBS中短暂洗涤,放置 在低温模具内,覆盖有OCT组织冷冻介质和卡扣 通过将低温模具放置在液氮顶部来冷冻
    3. 然后使用低温恒温器将冷冻切片肾切成10μm切片。
      1. 关于如何切割切片的一般指导,请参考Fischer等人(2008)所做的工作。
    4. 将待处理的样品在室温下解冻10分钟,然后用冰冷的100%丙酮在-20度固定10分钟。


    1. 将同种型孵育的载玻片用作阴性对照。
    2. 所有洗涤步骤在Coplin罐中进行。
    1. 切片在1×PBS中洗涤2×5分钟。
    2. 内源性过氧化物酶使用DAKO的赘水过氧化物酶封闭 ?根据制造商的说明在室温下封闭7分钟
    3. 切片在1×PBS中洗涤2×5分钟。
    4. 切片与山羊抗小鼠Rae-1γ抗体(1/100 在含有10%FBS和2.5%BSA的1×PBS中稀释)在4℃过夜
    5. 切片在1×PBS中洗涤2×5分钟。
    6. 根据制造商的说明书,将切片与山羊Histofine简单染色试剂盒在RT温育30分钟
    7. 切片在1×PBS中洗涤2×5分钟。
    8. 根据制造商的说明书,将切片在室温下用AEC +底物 - 色原体染色15-20分钟
    9. 通过用蒸馏轻轻冲洗载玻片来终止反应 来自洗瓶的水(不要将流体直接聚在纸巾上)
    10. 切片用苏木精复染3分钟。
    11. 通过将载玻片置于流动自来水下10分钟停止反应
    12. 将1滴水性封固剂置于每个载玻片上,小心地加入盖玻片以不形成气泡
    13. 将载玻片在室温下干燥1小时
    14. 通过定量测量色原沉积 通过使用已建立的方法进行免疫组织化学(参见B节 "图像分析")。
    15. 肾小球的图像(放大100倍) 在Leica显微镜(垂直Leitz DM RB)中获得 奥林巴斯(DP70)相机;图像文件保存在带标签的图像文件中 格式(抗体染色的图像称为EXP和对照 图像被称为CON??TROL)。

  2. 图像分析
    1. 在Photoshop中打开图片。
    2. 定义选框工具的大小参数。
      1. 双击选框图标。
      2. 以像素(200 x 200像素区域)定义面积。
      注意:该区域必须任意选择尽可能最小 区域,其中可以看见整个肾小球。然后用户必须 ?继续选择那些适合舒适的肾小球 ?区域(参见步骤B7)。相同的像素区域必须用于所有样本 每组。在我们的例子中,200×200像素区域被证明是 最适合。
    3. 使用选框工具标识要分析的区域。
    4. 存储在新文件中。
      1. 功能"编辑"选择"复制"。
      2. 功能"编辑"选择"新建文件"。
      3. 在新文件中选择"粘贴"。
      4. 功能"保存"为文件类型选择tiff。
    5. 将新文件存储在Matlab文件夹中。
    6. 打开Matlab。
      ?a = double(imread('EXP','tiff'));
      ?b = double(imread('CONTROL','tiff'));
      ?ea = norm(a(:,:,1))+ norm(a(:,:,2))+ norm ?eb = norm(b(:,:,1))+ norm(b(:,:2))+ norm ?et = abs(ea-eb);
      ?en = sqrt(et o2/N)
    7. 根据该方案,测定色原体/像素的量 ?通过在200×200像素区域中选择肾小球(25个肾小球/组) ?并减去数学能量(在步骤B6计算的EM) 对照载玻片(对照 - 未暴露于一抗) 的同源肾小球在实验载玻片上(EXP-暴露于 Rae-1抗体)。
      注意:使用了200 x 200像素区域,因为 它被证明是能够包括的区域大小 所有样品的肾小球以最可能的同源方式。这个大小 必须根据手边的样品进行调整。


图1.来自1岁的MRL/MpJ小鼠模型的肾中IHC Rae-1染色(红色)的实施例。在10x。A阴性对照。 B.与Rae-1抗体孵育的载玻片

图2.在100x放大率下从肾小球提取的实施例200×200像素区域。 A.来自阴性对照玻片的肾小球; B.来自Rae-1孵育玻片的肾小球。

图3.MRL小鼠肾小球中Rae-1γ染色的定量使用双尾,不配对的学生t检验(平均6SD; n = 5 /组; ** P, 0.0001; * P,0.0025)。


  1. 这种技术允许量化绝对量 Rae-1抗体色原体的每个像素在肾小球的健康和 患病小鼠。然而,这种技术不限于 肾小球或Rae-1抗体,因为它可以根据需要进行修饰 ?任何细胞中任何抗体的染色强度的测定 ?区域或结构。
  2. 所有样品(包括阴性对照)都是至关重要的 处理和同时分析,以便最小化变化 染色结果。


这项工作部分得到了西班牙经济和竞争力部(SAF-2008-00471和SAF-2011-23639; DFB),马德里地区政府(CCG08-CSIC/SAL-3451; DFB )和西班牙卫生,社会服务和平等部,Carlos III健康研究所合作研究专题网络计划:炎症和风湿病研究网络(RIER; RD08/0075/0015; DFB)和西班牙国家生物银行网络/0076/00101到FJD)。 R. S.得到了来自西班牙经济和竞争力部的FPI授职奖学金的支持。我们要感谢Matkowskyj等人(2000)的工作,该工作是该协议的基础。


  1. Fischer,A.H.,Jacobson,K.A.,Rose,J。和Zeller,R。(2008)。 冷冻切片组织 CSH Protoc 2008:pdb prot4991。
  2. Matkowskyj,K.A.,Schonfeld,D。和Benya,R.V。(2000)。 通过使用市售软件photoshop和matlab测量累积信号强度的定量免疫组织化学。 J Histochem Cytochem 48(2):303-312。
  3. Spada,R.,Rojas,J.M.,Perez-Yague,S.,Mulens,V.,Cannata-Ortiz,P.,Bragado,R.and Barber,D.F。 NKG2D配体在狼疮性肾炎中的过度表达与肾脏中NK细胞活性和分化增加有关,但与.Leukoc Biol 97(3):583-598。
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引用:Spada, R. and Barber, D. F. (2016). Quantification of Rae-1 Staining Intensity in Glomeruli. Bio-protocol 6(5): e1754. DOI: 10.21769/BioProtoc.1754.