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Resin-embedded Thin-section Immunohistochemistry Coupled with Triple Cellular Counterstaining
树脂包埋薄层免疫组织化学法联合三重细胞复染   

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参见作者原研究论文

本实验方案简略版
Molecular Plant
Feb 2015

Abstract

This protocol was developed to study protein localisation within the vascular bundles of developing tomato fruit however, it can be applied to any resin embedded plant tissue. The vascular bundle is comprised of many different cells that all have unique properties. The mature sieve elements are enucleated and contain sieve plates that comprise of callose. This method has utilised these properties of the sieve element by combining immunohistochemistry for cell wall invertase with counterstaining of aniline blue for callose, DAPI for nucleus and cell structure is shown with the final staining of the cell wall using calcofluor white. It must be noted that when following this protocol, it is vital for the sections to be flat and fixed to the slide with gelatine so cover slip removal does not move the sample section. This protocol will be applicable to all plant tissues and provides additional evidence of the protein localisation within the cell by conducting a counterstaining procedure.

Keywords: Immunolocalisation (免疫定位), Microscopy (显微镜检查), Staining (染色), Phloem (韧皮部)

Background

Immunolocalisation has long been a method used to study the localisation of proteins within tissue. This protocol focused on, not only localising the proteins of interest but also the molecular structures that were in surrounding tissue. It is believed that the mature sieve elements are enucleated and have abundant callose deposition within the sieve plate. Therefore, counterstaining procedures were applied in order to represent these biological phenomena. As the proteins of interest in this study were also thought to be localised within the apoplast further counterstaining was applied showing co-labelling of the protein and the cell wall.

Materials and Reagents

  1. Size 000 gelatine capsules (ProSciTech, catalog number: RL039 )
  2. Razor blade
  3. Microscope slide (Livingstone, catalog number: 7107-PPN )
  4. 22 x 50 mm, 0.17 mm thick coverslip (Fisher Scientific, catalog number: 12-543C )
  5. Tomato flowers 2 Days Before Fertilization (DBF) and 2 Days After Fertilization (DAF) harvested from glasshouse-grown cv. Moneymaker tomato plants
  6. 50 mM PIPES (Sigma-Aldrich, catalog number: F6757 )
  7. AR grade EtOH (Sigma-Aldrich, catalog number: 32205 )
    Note: This product has been discontinued.
  8. ddH2O
  9. LR white resin (ProSciTech, catalog number: C023 )
  10. Gelatine (Sigma-Aldrich, catalog number: G9391 )
  11. Cell wall invertase (LIN5) purified polyclonal primary antibody – produced in rabbit (Mimmitopes – custom made)
  12. Inhibitor of invertase (INH) purified polyclonal primary antibody – produced in rabbit (Mimmitopes – custom made)
  13. TBST (Sigma-Aldrich, catalog number: T9039 )
  14. Secondary anti-rabbit IgG fluorescein isothiocyanate (FITC) (Sigma-Aldrich, catalog number: F9887 )
  15. Anti-Rabbit IgG (whole molecule)-FITC antibody produced in goat (Sigma-Aldrich, catalog number: F6005 )
    Note: This product has been discontinued.
  16. Aniline blue (0.1% in ddH2O) (Sigma-Aldrich, catalog number: B8563 )
  17. Mowiol-phenylenediamine (mowiol) (Sigma-Aldrich, catalog number: 10852 )
  18. 4’,6-diamidino-2-phenylindole (DAPI) (1:500) (Sigma-Aldrich, catalog number: D9542 )
  19. Calcofluor white (0.1% in ddH2O) (Sigma-Aldrich, catalog number: F3543 )
  20. 2% paraformaldehyde
  21. Glutaraldehyde
  22. CaCl2
  23. Tris
  24. NaN3
  25. Bovine serum albumin (BSA)
  26. Fixing solution (see Recipes)
  27. Blocking buffer (see Recipes)

Equipment

  1. Rotator
  2. Dissecting microscope or magnifying glass
  3. Reichert Ultracut E microtome (Reichert, model: 701704 )
  4. DiATOME Histo Knife, Diamond, 45°, 4.0-4.9 mm (ProSciTech, catalog number: UH45-40 )
  5. Fume hood
  6. Beaker
  7. Axio Scope.A1 epifluorescence compound microscope (ZEISS, model: Axio Scope.A1 )
  8. Emission FITC filter (50-490 nm excitation, long pass 515 nm)
  9. Emission UV filter (365 nm excitation, short pass 420 nm)
  10. AxioCam digital camera (ZEISSTM) or equivalent

Software

  1. AxioVision V4.8 software
  2. Adobe Bridge CS4 software
  3. Abode Photoshop CS4 software

Procedure

  1. Sample preparation
    1. Harvest whole tomato flowers 2 DBF and 2 DAF from glasshouse-grown tomato plants and placed on ice for 4 h in fixing solution under vacuum (300 mm Hg) to remove air bubbles and submerge the entire flower.
    2. Wash flowers 3 x 10 min in 50 mM PIPES and store overnight at 4 °C.
    3. Was a further 3 x 10 min in ddH2O preceding ethanol dehydration.
    4. Ethanol dehydration series is progressed 10-100% in 10% increments whilst flowers are submerged in 5 ml of solution for 1 h per increment at 4 °C up to 70% then store tissue overnight.
    5. The following day remaining increments 80% and 90% are applied for 1 h at RT. Once in 100% ethanol, flowers are washed twice in 100% at 1 h increments before again being stored overnight.
    6. LR white resin is diluted with ethanol and infiltration series conducted as above with LR white concentrations starting at 10% through to 100% on a rotator at RT.
    7. Once samples are in 100% LR white resin, change with 5 ml fresh 100% LR white solution every 2-3 days for a period of three weeks.
    8. Size 000 gelatine capsules are 1/3 filled with LR white resin and partly polymerised at 60 °C (~12 h). Flowers are then placed into middle of each capsule with petals pointing up, on the partly polymerised resin and filled with LR white resin.
    9. The gelatine capsules are sealed and placed in 60 °C oven for 48 h until resin is totally polymerised.
    10. Hardened capsules are then trimmed with a razor blade around each flower sample under a dissecting microscope leaving 1 mm resin around all surfaces.
    11. The trimmed blocks are securely fastened onto a Reichert Ultracut E microtome and 1 µm thick sections cut using 45 Diamond DiATOME Histo Knife, 4.0-4.9 mm.
    12. Sections are floated onto water at RT within the knife boat before being transferred to a drop of ddH2O on a gelatine coated LIVINGSTONETM microscope slide.
    13. Three sections are placed on each slide and dried on a laboratory warming tray at 45 °C in fume hood under a beaker with a chloroform soaked cotton wool bud attached to the underside until water drop evaporated and the section flattens onto the slide. 
       
  2. Immunolabeling
    1. 1 ml of blocking buffer is pipetted onto slide for 2 h (a hydrophobic pen can be used to prevent leakage). Excess solution is dried off the edges of each slide.
    2. Thereafter, 200 µl of 1:100 LIN5 or INH antibody, diluted in TBST, is pipetted to each slide and incubated for 1 h at RT.
    3. Slides are then washed 3 x 20 min with TBST. Once again excess solution is blotted from slide with lint free paper (without touching sections) and secondary anti-rabbit IgG fluorescein isothiocyanate (FITC) conjugated antibody, produced in goat is applied at a dilution of 1:200 in TBST incubated for 1 h at RT as above in dark conditions.
    4. Slides are washed as above with successive wash steps in TBST and then ddH2O (Figure 1A).

  3. Counterstaining
    1. A novel counterstaining procedure was developed to ascertain cellular localisation of LIN5 and INH.
    2. Following immunolabelling, 200 µl of aniline blue is applied to the washed sections and incubated for 20 min in dark conditions.
    3. Excess aniline blue is washed off by slowly pipetting 5 ml TBST over sections while holding at a 45° and then sections are mounted in 20 μl mowiol-phenylenediamine (mowiol) before a 22 x 50 mm long coverslip is applied.
    4. Sections are viewed under a ZEISSTM AxioScope. A1 epifluorescence compound microscope. Immunolabelling is viewed using 450-490 nm excitation, long pass 515 nm emission FITC filter set with at 100x objective under oil emersion (Figure 1A).
    5. Once immunolabelling signal is detected, rapid switching to 365 nm excitation, short pass 420 nm emission UV filter set is achieved to detect fluorescent callose upon binding to aniline blue, as the latter fades rapidly. The image is captured on a ZEISSTM AxioCam digital camera using AxioVision V4.8 software (Figure 1B). Filter set is returned to FITC and another image was taken of immunolabelling in the same target area (Figure 1A).
    6. When removing each slide from the microscope stage, care must be taken to keep the stage in the same position and to note the orientation of the slide.
    7. Once slide had been removed, the coverslip was slid off the slide with extreme care and discarded.
    8. Sections are washed with TBST as above until all excess mowiol is removed and 200 µl of 1:500 4’,6-diamidino-2-phenylindole (DAPI) is applied to locate the nucleolus and incubated at RT for 30 sec.
    9. Excess DAPI is removed with TBST and slides re-mounted as described above.
    10. The target area is re-acquired and aligned whilst being viewed under the FITC filter and compared to the previously captured image (Figure 1A) before being switching to long pass 420 nm emission UV filter set and capturing the image of DAPI stained cells (Figure 1C).
    11. As with DAPI counterstaining procedure, Calcofluor white staining proceeds as above to label cell wall cellulose (Figure 1D).
    12. All images are then exported and overlayed using Abode Photoshop CS4 software package (Figure 1E).


      Figure 1. Resin embedded tomato flower section of the placental vascular region 2 DBF. A. Immunolabelling of LIN5 antisera on the cell wall of the sieve element; B. Counterstaining of same section with aniline blue showing the sieve plate at anterior and posterior ends of the sieve element; C. Further counterstaining of same section with DAPI showing nucleus of surrounding cells and no nucleus within sieve elements; D. False coloured red image of cell walls stained with calcofluor white; E. Overlay of A, B, C and D. [zoom] overlay showing LIN5 localisation within the cell wall of the sieve element. All images acquired on a ZEISS compound fluorescence microscope at 100x objective. Scale bars = 10 µm. labelled cell types = (N) nucleus, (CC) companion cell, (PP) phloem parenchyma, (SP) sieve plate and (SE) sieve element. (This image was reproduced from Palmer et al., 2015.)

Data analysis

Images sequences were imported into Adobe Bridge CS4 software before being loaded into Adobe Photoshop CS4 software and overlaid with opacity and intensity levels adjusted.

Notes

Gelatine coated slides need to be well coated to ensure stability of section on the microscope slide during removal of coverslip for optimal overlaying of images acquired.

Recipes

  1. Fixing solution
    50 mM PIPES, pH 6.8
    2% paraformaldehyde
    1.5% glutaraldehyde
    2 mM CaCl2
  2. Blocking buffer
    20 mM Tris, pH 7
    150 mM NaCl
    0.2% NaN3
    2% bovine serum albumin

Acknowledgments

The condensed version of this method was originally published in Palmer et al., 2015.
This work was collectively supported by the National Science Foundation of China (Grant number 30425043 to Y.L.R.) and Australia Research Council (ARC DP110104931 to Y.L.R. and DP120104148 to Y.L.R. and J.W.P.).

References

  1. Palmer, W. M., Ru, L., Jin, Y., Patrick, J. W. and Ruan, Y. L. (2015). Tomato ovary-to-fruit transition is characterized by a spatial shift of mRNAs for cell wall invertase and its inhibitor with the encoded proteins localized to sieve elements. Mol Plant 8(2): 315-328.

简介

该方案被开发用于研究番茄果实的维管束内的蛋白质定位,但可应用于任何树脂嵌入植物组织。 血管束由许多不同的细胞组成,它们都具有独特的特性。 成熟的筛分元素被去核并含有包含胼lose质的筛板。 该方法通过将细胞壁转化酶的免疫组织化学与苯胺蓝复染以胼cal质结合使用筛选元件的这些性质,细胞核的DAPI和细胞结构用细胞壁的最终染色显示为calcofluor白色。 必须注意的是,按照本协议,这些部分至关重要的是使用明胶平坦固定在滑块上,因此盖板滑移不会移动样品部分。 该方案将适用于所有植物组织,并通过进行复染试验,提供细胞内蛋白质定位的其他证据。
免疫定位一直是用于研究蛋白质在组织内的定位的方法。 该方案不仅局限于感兴趣的蛋白质,还涉及周围组织中的分子结构。 据信成熟的筛子元素被去核并且在筛板内具有丰富的胼cal质沉积。 因此,为了代表这些生物学现象,应用了复染程序。 由于本研究中感兴趣的蛋白质也被认为是定位在质外体中,进一步的复染显示蛋白质和细胞壁的共标记。

关键字:免疫定位, 显微镜检查, 染色, 韧皮部

材料和试剂

  1. 尺寸000明胶胶囊(ProSciTech,目录号:RL039)
  2. 剃刀刀片
  3. 显微镜幻灯片(Livingstone,目录号:7107-PPN)
  4. 22 x 50 mm,0.17 mm厚的盖玻片(Fisher Scientific,目录号:12-543C)
  5. 番茄花2天施肥(DBF)和2天后施肥(DAF)从温室栽培的cv。 Moneymaker番茄植物
  6. 50mM PIPES(Sigma-Aldrich,目录号:F6757)
  7. AR级EtOH(Sigma-Aldrich,目录号:32205)
    注意:本产品已停产。
  8. ddH 2 O
  9. LR白色树脂(ProSciTech,目录号:C023)
  10. 明胶(Sigma-Aldrich,目录号:G9391)
  11. 细胞壁转化酶(LIN5)纯化的多克隆抗体 - 兔产生(Mimmitopes - 定制)
  12. 转化酶(INH)纯化的多克隆抗体 - 兔产生的抑制剂(Mimmitopes - 定制)
  13. TBST(Sigma-Aldrich,目录号:T9039)
  14. 次级抗兔IgG荧光素异硫氰酸酯(FITC)(Sigma-Aldrich,目录号:F9887)
  15. 山羊中产生的抗兔IgG(全分子)-FITC抗体(Sigma-Aldrich,目录号:F6005)
    注意:本产品已停产。
  16. 苯胺蓝(在ddH 2 O中为0.1%)(Sigma-Aldrich,目录号:B8563)
  17. (Mowiol)(Sigma-Aldrich,目录号:10852)
  18. 4',6-二脒基-2-苯基吲哚(DAPI)(1:500)(Sigma-Aldrich,目录号:D9542)
  19. Calcofluor白色(在ddH 2 O中为0.1%)(Sigma-Aldrich,目录号:F3543)
  20. 2%多聚甲醛
  21. 戊二醛
  22. CaCl 2
  23. Tris
  24. NaN <3>
  25. 牛血清白蛋白(BSA)
  26. 固定解决方案(请参阅配方)
  27. 阻塞缓冲区(见配方)

设备

  1. 旋转器
  2. 解剖显微镜或放大镜
  3. Reichert Ultracut E切片机(Reichert,型号:701704)
  4. DiATOME Histo刀,钻石,45°,4.0-4.9mm(ProSciTech,目录号:UH45-40)
  5. 通风柜
  6. 烧杯
  7. Axio Scope.A1 epiflourescence复合显微镜(ZEISS,型号:Axio Scope.A1)
  8. 发射FITC滤光片(50-490nm激发,长波长515nm)
  9. 发射UV滤光片(365nm激发,短波420nm)
  10. AxioCam数码相机(ZEISS TM )或等效的

软件

  1. AxioVision V4.8软件
  2. Adobe Bridge CS4软件
  3. Abode Photoshop CS4软体

程序

  1. 样品制备
    1. 从番茄植物番茄植物中收获全番番茄2朵DBF和2朵DAF,并在真空(300毫米汞柱)下,在冰上固定溶液4小时,以除去气泡并淹没整个花朵。
    2. 在50mM PIPES中洗涤3×10分钟,并在4℃下储存过夜。
    3. 在乙醇脱水之前,在ddH 2 O 2中再进一步3×10分钟。
    4. 乙醇脱水系列以10%的增量进行10-100%,而花被浸泡在5ml溶液中,每4℃加入1小时,最高达70%,然后将组织储存过夜。
    5. 第二天剩余的增量为80%,90%在室温下施用1小时。一旦在100%乙醇中,花以100%的速率洗涤两次,每1小时增量一次,然后再次存放过夜
    6. 用乙醇稀释LR白色树脂,并在室温下在旋转器上进行如上进行的LR白色浓度的10%至100%的渗透系列。
    7. 一旦样品在100%LR白色树脂中,每2-3天更换5ml新鲜的100%LR白色溶液,持续三周。
    8. 尺寸为000的明胶胶囊填充有LR白色树脂,并在60℃(〜12小时)下部分聚合。然后将花瓣放置在每个胶囊的中间,花瓣朝上,部分聚合的树脂上充满LR白色树脂。
    9. 将明胶胶囊密封并置于60℃烘箱中48小时,直到树脂完全聚合。
    10. 然后用解剖显微镜在每个花样品周围用剃刀刮刀硬化胶囊,在所有表面上留下1mm树脂。
    11. 修剪的块被牢固地固定在Reichert Ultracut E切片机上,并且使用45钻石DiATOME Histo刀(4.0-4.9mm)切割1μm厚的切片。
    12. 切片在刀片舟皿内在RT上漂浮在水上,然后在明胶包被的LIVINGSTONE TM超声显微镜载玻片上转移到一滴ddH 2 O。
    13. 将三个部分放置在每个载玻片上,并在实验室升温托盘上在45℃的通风橱中在烧杯下干燥,氯仿浸泡的棉絮芽附着在下面,直到水分蒸发,并且该部分平坦化到载玻片上。 br />  
  2. 免疫标记
    1. 将1ml阻塞缓冲液吸移到载玻片上2小时(可以使用疏水笔来防止泄漏)。过量溶液从每个载玻片的边缘干燥。
    2. 此后,将200μl在TBST中稀释的LIN5或INH抗体移液至每个载玻片并在室温下孵育1小时。
    3. 然后用TBST洗涤3×20分钟的幻灯片。再次用无绒纸(无接触切片)从载玻片中吸收过量溶液,并在TBST中以1:200的稀释度施用在山羊中产生的第二抗兔IgG荧光素异硫氰酸荧光素(FITC)缀合的抗体,孵育1小时, RT在黑暗条件下如上。
    4. 在TBST中随后洗涤步骤,然后用ddH 2 O(图1A)洗涤如上所述的幻灯片。

  3. 复印
    1. 开发了一种新的复染方法,以确定LIN5和INH的细胞定位。
    2. 免疫标记后,将200μl苯胺蓝施用于洗涤的部分,并在黑暗条件下孵育20分钟。
    3. 在保持45°的情况下,通过缓慢移液5 ml TBST,然后将切片安装在20μl马可酯 - 苯二胺(马可酚)中,然后涂上22 x 50 mm长的盖玻片,洗掉过量的苯胺蓝。
    4. 部分由ZEISS TM AxioScope查看。 A1 epiflourescence复合显微镜。使用450-490nm的激发,长通515nm发射FITC滤光片,以100x物镜在油浸液下观察免疫标记(图1A)。
    5. 一旦检测到免疫标记信号,就可快速切换到365nm激发,通过420nm发射紫外滤光器组,以便在结合苯胺蓝时检测荧光胼filter质,随后者快速消失。使用AxioVision V4.8软件在ZEISS TM AxioCam数码相机上捕获图像(图1B)。过滤器组返回到FITC,并在同一目标区域拍摄另一张免疫标记图像(图1A)。
    6. 当从显微镜平台上移除每张幻灯片时,必须注意将台架保持在相同位置,并注意幻灯片的方向。
    7. 一旦幻灯片被移除,盖玻片就会非常小心地从幻灯片上滑下来并被丢弃。
    8. 切片用如上所述的TBST洗涤,除去除去所有过量的马索醇,并加入200μl1:500 4',6-二脒基-2-苯基吲哚(DAPI)以定位核仁,并在室温下孵育30秒。
    9. 用TBST去除过量的DAPI,如上所述重新安装滑块。
    10. 在FITC滤波器之前,重新获取和对准目标区域,并将其与先前捕获的图像进行比较(图1A),然后切换到长通道420nm发射UV滤光片组并捕获DAPI染色细胞的图像(图1C )。
    11. 与DAPI复染方法一样,Calcofluor白色染色如上所述进行标记细胞壁纤维素(图1D)。
    12. 然后使用Abode Photoshop CS4软件包导出所有图像并重叠(图1E)。


      图1.胎盘血管区2 DBF的嵌入番茄花的树脂。A.在筛孔的细胞壁上免疫标记LIN5抗血清; B.将具有苯胺蓝色的相同部分的复染显示在筛子元素的前端和后端的筛板; C.用DAPI进一步复染同一部分,显示周围细胞的核,并且筛孔内没有细胞核; D.用calcofluor白色染色的细胞壁的虚假红色图像; E.叠加A,B,C和D. [缩放]覆盖,显示在筛元件的细胞壁内的LIN5定位。在ZEISS复合荧光显微镜上获得的所有图像均为100倍物镜。刻度棒=10μm。标记细胞类型=(N)核,(CC)伴侣细胞,(PP)韧皮实质,(SP)筛板和(SE)筛分元素。 (此图像由Palmer等人,2015年出版)

数据分析

图像序列被导入到Adobe Bridge CS4软件中,然后被加载到Adobe Photoshop CS4软件中,并且覆盖了不透明度和强度水平的调整。

笔记

明胶涂层的载玻片需要良好的涂层,以确保在清除盖玻片时切片在显微镜载玻片上的稳定性,以获得获得的图像的最佳覆盖。

食谱

  1. 固定解决方案
    50 mM PIPES,pH 6.8
    2%多聚甲醛
    1.5%戊二醛
    2mM CaCl 2
  2. 阻塞缓冲区
    20mM Tris,pH 7
    150 mM NaCl
    0.2%NaN 3
    2%牛血清白蛋白

致谢

该方法的精简版本最初发布于Palmer等人,2015年。
这项工作得到了中国国家科学基金会的统一支持(授权号码 30425043 和澳大利亚研究委员会( ARC DP110104931 到YLR和 DP120104148 到YLR和JWP)。

参考文献

  1. Palmer,WM,Ru,L.,Jin,Y.,Patrick,JW and Ruan,YL(2015)。 
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Palmer, W. M., Patrick, J. W. and Ruan, Y. (2017). Resin-embedded Thin-section Immunohistochemistry Coupled with Triple Cellular Counterstaining. Bio-protocol 7(7): e2052. DOI: 10.21769/BioProtoc.2052.
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