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Imaging Thick Lymph Node Tissue Sections
淋巴结组织厚切片的成像技术   

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Abstract

Our protocol describes a simple procedure for imaging thick lymph node sections by 2-photon microscopy. Lymph nodes are sectioned using a vibratome (vibrating microtome) to produce slices of tissue that can then be stained with fluorescently labeled antibodies. The thick tissue sections (150-200 μm depth) allow for the detection of cell clustering that is typically under-represented in thin sections (10-20 μm) used for conventional confocal microscopy. Application of 2-photon microscopy facilitates imaging through the thick volume of the vibratome sections. In combination with automated image processing software, a thick lymph node cross-section image also facilitates quantitation of cellular events within a relatively large area of the tissue, thus providing a clearer picture on the spatial distribution of cellular events of interest (e.g., T cell clustering). This method can also readily be applied to other tissues, such as the spleen or skin.

Keywords: 2-photon microscopy(双光子显微镜), Lymph nodes(淋巴结), T lymphocytes(T淋巴细胞), Vibratome(振动切片机), Immune response(免疫反应)

Materials and Reagents

  1. Razor blades, single edge, No.9 (0.22 mm) (VWR International, catalog number: 55411-050 )
  2. Greiner CELLSTAR® 24-well flat-bottom plates (Sigma-Aldrich, catalog number: M8812 )
  3. 96-well round-bottom plates (Corning, catalog number: 3788 )
  4. Durapore tape (3M, catalog number: 1538-1 )
  5. SuperfrostTM Plus and ColorFrostTM glass slides (25 x 75 x 1.0 mm) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4951PLUS4 )
  6. Coverslips, No.1.5 (ProSciTech, catalog number: G425-2460 )
  7. Agarose (Sigma-Aldrich, catalog number: A6013-250G )
  8. Vetbond tissue adhesive (3M, catalog number: 1469SB )
  9. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  10. Protein block (Dako, catalog number: X0909 )
  11. Normal donkey serum (NDS) (Jackson ImmunoResearch, catalog number: 017-000-121 )
  12. Prolong gold antifade reagent (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: P36930 )
  13. Antibodies [e.g., pacific blue anti-B220, RA3-6B2 (Biolegend, catalog number: 103230 ); goat anti-CD69 (R&D Systems, catalog number: AF2386 )]
  14. Sodium periodate (NaIO4) (Sigma-Aldrich, catalog number: 311448 )
  15. Sodium dihydrogen orthophosphate, monohydrate (NaH2PO4·H2O) (monobasic) (VWR, catalog number: 97062-412 )
  16. Di-sodium hydrogen orthophosphate, anhydrous (Na2HPO4) (dibasic) (Ajax Finechem, catalog number: 621-500G )
  17. L-lysine (Sigma-Aldrich, catalog number: L5501 )
  18. Gelatin from porcine skin (Sigma-Aldrich, catalog number: G1890-100G )
  19. Glycerol for molecular biology (≥ 99%) (Sigma-Aldrich, catalog number: G5516 )
  20. 16% paraformaldehyde aqueous solution (Electron Microscopy Sciences, catalog number: 30525-89-4 )
  21. Periodate-lysine-paraformaldehyde (PLP) fixative (see Recipes)
  22. P-buffer (see Recipes)
  23. L-lysine (see Recipes)
  24. 5% gelatin/glycerol mix (see Recipes)

Equipment

  1. Vibratome (Leica, model: VT1200S )
  2. Dumont No.5 forceps (Fine Science Tools, catalog number: 11251-10 )
  3. Scissors, straight, sharp (Roboz, catalog number: RS-6752 )
  4. Soft bristled paint brush (e.g., size 2/0)
  5. Microscope with tunable Coherent Chameleon Ti:Sa laser (ZEISS, model: LSM 710 NLO )
    Note: This product has been discontinued (Replaceable item, e.g., ZEISS, model: LSM 800 ).
  6. Scale for weighing 1 g
  7. Microwave

Software

  1. Imaris, Bitplane (http://www.bitplane.com/)

Procedure

  1. Harvest lymph nodes (LNs) and place in 2 ml of PLP or 2% paraformaldehyde (PFA) at 4 °C for fixation.
    Notes:
    1. This step is optional but crucial for preserving cellular morphology, but may compromise certain antibody staining. One example we have tested is anti-XCR1 (clone: ZET) where fixation abrogates staining. PLP is preferable for preserving fine cellular morphology, such as cell dendrites.
    2. All procedures involving animals must be performed according to approved institutional, regional or national protocols (as applicable) by appropriately trained and certified personnel.
  2. After fixation (4-6 h for small LNs, and up to 12 h for larger tissues, such as swollen LN after inflammation), transfer tissues into 2 ml of cold PBS and keep on ice.
  3. Embed tissues into agarose gel:
    1. Pour warm 2% agarose in PBS (boiled then cooled under running tap water until warm to touch) into wells in 24-well plates;
    2. Embed tissues into wells loaded with warm agarose and in desired orientation, Figure 1 (Each LN should touch the bottom of the well and will later be sliced parallel to this plane);
    3. Make sure the plate is level and leave on ice to allow agarose to set.


      Figure 1. Embedding LN in agarose gel. Pour warm agarose into wells, embed LN into agarose and adjust to desired orientation (left). Allow agarose to set (right).

  4. Meanwhile, start up the vibratome and calibrate the cutting blade as per manufacturer’s instructions. Prepare an ice bath and fill the buffer tray with cold PBS, Figure 2.


    Figure 2. Setting up vibratome. Insert blade and calibrate, prepare ice bath, and fill buffer tray with cold saline buffer.

  5. Remove the agarose block containing the LN with a pair of forceps and carefully trim into cuboid shape with a razor blade, Figure 3.
  6. Apply 1-2 strips of 3M Durapore tape to the specimen holder. Attach the trimmed LN/agarose block on the specimen holder (with the LN near the top of the block) using a drop of Vetbond adhesive (Figure 3, right).
    Note: Cyanoacrylate glue as recommended by the manufacturer is preferred. However, if Vetbond is used, wait ~1 min after tissue block is attached for the glue to dry.


    Figure 3. Mounting LN/agarose block. Remove set agarose gel from well (left), trim with a razor blade into cuboid shape (middle) and mount the block on to the specimen holder with a small amount of glue (right).

  7. Mount the specimen holder onto vibratome for cutting. The LN should be aligned such that it is sectioned longitudinally as we find that tissue quality is better preserved (Figure 4).
  8. Set up the vibratome to ensure appropriate cutting speed and thickness. Typically, we use ~0.03 mm/s blade speed with ~1.6 mm amplitude, and ~200-250 μm thickness for best preservation of LN structural integrity.
  9. Begin with the vibratome blade just above the agarose surface, using brush as guide, and begin cutting via automated single or continuous sectioning. Discard the first slice.
  10. Transfer cut slices (with a No.5 forcep and soft bristled brush) into wells in a 96-well round-bottom plate filled with cold PBS, Figure 4. Take particular care to grip only the agarose part of the slice so as not to disrupt the LN tissue.


    Figure 4. Handling vibratome slices. Once an LN/agarose slice has been cut (left), use a pair of fine forceps and a brush to retrieve the slice from the buffer tray (middle) and leave it in cold PBS. For reference, the slice should appear relatively intact with minimal disruption to the tissue (right).

  11. Prepare a 96-well round-bottom plate for antibody staining by filling each row with reagents to be used in sequential steps for antibody staining (Figure 5):
    Row 1: Cold PBS (where LN slices are initially placed)
    Row 2: Protein block
    Row 3: Primary antibodies (~70 μl per well)
    Row 4: PBS (Wash #1)
    Row 5: PBS (Wash #2)
    Row 6: Secondary antibodies (~70 μl per well)
    Row 7: PBS (Wash #1)
    Row 8: PBS (Wash #2)
  12. Transfer the LN slices down each column after each incubation step.
  13. Incubate in protein block for 1-2 h.
  14. Transfer into the well containing the primary antibodies (diluted in 2.5% NDS) and incubate at 4 °C for 8 h or overnight.
    Note: We have tested Pacific Blue-conjugated anti-B220 (dilution 1:200) and unconjugated anti-CD69 (dilution 1:50). It should be noted that each antibody batch should be individually titrated for optimal results.
  15. Transfer to the first wash buffer (PBS) well for 20-30 min, followed by the second well containing wash buffer (PBS).
  16. Transfer into the well containing the secondary antibodies (diluted in 2.5% NDS) and incubate at 4 °C for 8 h or overnight.
  17. Transfer to the first wash buffer (PBS) well for 20 min, followed by the second well containing wash buffer (PBS).


    Figure 5. Plate setup for antibody staining. Each well contains a LN slice that is transferred down each column for antibody labeling and washing steps.

  18. Place LN slices on glass microscope slides (up to 4 LN slices per slide). Arrange them in desired orientation.
  19. Use 5% gelatin/glycerol in PBS as mounting media and carefully place a coverslip over the tissues such that the edges are sealed.
  20. Slides are now ready for two-photon imaging:
    Acquisition parameters differ and are dependent upon the desired quality and fluorophore configuration (see Notes).
    In our experiments, we use: Zeiss LSM710 NLO with tunable Coherent Chameleon Ti:Sa laser Zeiss 20x/1.0 NA water immersion lens.
    Resolution: 512 x 512
    Slice interval: 2.5 μm
    Zoom: ~1.5x
    Depth-adjusted correction for laser power and gain settings to reduce excessive laser excitation of the tissue, which can lead to photobleaching and phototoxicity.
    Whole LN cross-sections are acquired using the tile-scanning function of the Zeiss Zen software. An example is shown in Figure 6.
  21. Post-acquisition processing can be performed on dedicated image processing software (e.g., Bitplane Imaris) to allow quantitation of cells (Spots function) and export of statistics. Additionally, Imaris XTension functions or customized MATLAB script can be used for further analysis.

Representative data



Figure 6. Example image of LN slice acquired using 2-photon microscopy. Shown is a 150 μm slice of mouse inguinal LN populated by CellTrace Violet-labelled CD4 T cells (green), CellTracker Deep Red-labelled CD8 T cells (white) and TRITC-labelled migratory DC (red). Cells were labelled with CellTrace or CellTracker dyes as per the manufacturer protocols (Thermo Fisher Scientific). LN sections were then stained with Pacific Blue anti-B220 (blue). Also in blue was collagen delineating the LN capsule illuminated via second-harmonic generation. Image was acquired using Zeiss LSM710 NLO at dual wavelengths (800 nm for excitation of CellTrace Violet, CellTracker Deep Red and Pacific Blue, and 880 nm for TRITC excitation) and at 512 x 512 resolution in 8 x 4 tiles for a 3 x 1.5 mm LN cross-section. Image was then processed using Imaris (Bitplane). Autofluorescence and spectral crossover were removed using Channel Arithmetic function in Imaris XTension.

Notes

  1. The best tissue quality can be obtained with slower cutting speed. We find that 0.03 mm/s slices the tissue with minimal disruption. However, if deemed acceptable, cutting speed can be increased to save time.
  2. Prolong Gold mounting media can also be used in conjunction with gelatin/glycerol to take advantage of their antifade properties: place small drops of Prolong Gold on top of tissue slices, and gelatin/glycerol mix in the empty area of the slide. This should allow Prolong Gold to cover the tissue slices while allowing the gelatin/glycerol mixture to effectively seal off the edges of the slide.
  3. Although a confocal microscope can be used to acquire images of vibratome slices (we have achieved up to 40 μm depth), it could not image deep enough to take full advantage of the slice’s thickness. A 2-photon microscope is preferable for imaging up to 150-200 μm depth.
  4. Depending on the fluorophore combination, dual 2-photon excitation wavelengths may be used to acquire fluorescence with distinct excitation cross-section (e.g., use 800 nm for AlexaFluor dyes and 920 nm for EGFP/DsRed).
  5. Certain fluorophores are rapidly bleached by two-photon excitation and should be carefully considered. For example, we found that antibody conjugated to AlexaFluor 647 to be a poor choice for such purpose. In contrast, AlexaFluor 594 is relatively photostable for two-photon imaging.

Recipes

  1. Periodate-Lysine-Paraformaldehyde fixative
    For 10 ml solution:
    3.75 ml 0.1 M P-buffer (pH 7.4)
    3.75 ml 0.2 M L-lysine (pH 7.4)
    0.0213 g NaIO4
    2.5 ml 4% paraformaldehyde
    Note: Prepared fresh for each use.
  2. P-buffer, 0.1 M
    For 200 ml solution:
    19 ml 0.2 M NaH2PO4 (monobasic)
    81 ml 0.2 M Na2HPO4 (dibasic)
    100 ml H2O
    Adjust pH to 7.4
    Store at 4 °C
  3. L-lysine, 0.2 M
    For 100 ml solution:
    2.92 g L-lysine
    100 ml 0.1 M P-buffer
    Adjust pH to 7.4
    Store at 4 °C
  4. 5% gelatin/glycerol mix
    For 20 ml solution:
    1 g gelatin from porcine skin
    1 ml glycerol for molecular biology, ≥ 99%
    20 ml PBS
    Mix by inverting tube and leave in a 37 °C water bath to prevent hardening of gel
    Note: Prepared fresh for each use.

Acknowledgments

This protocol was adapted from our publication (Hor et al., 2015). This work was supported by the National Health and Medical Research Council, and by the Australian Research Council.

References

  1. Hor, J. L., Whitney, P. G., Zaid, A., Brooks, A. G., Heath, W. R. and Mueller, S. N. (2015). Spatiotemporally distinct interactions with dendritic cell subsets facilitates CD4+ and CD8+ T cell activation to localized viral infection. Immunity 43(3): 554-565.

简介

我们的协议描述了一个简单的程序,通过双光子显微镜成像厚淋巴结切片。 使用vibratome(振动切片机)切割淋巴结以产生组织切片,然后可用荧光标记的抗体染色。 厚组织切片(150-200μm深度)允许检测细胞聚集,其通常在用于常规共聚焦显微镜的薄切片(10-20μm)中表现不足。 双光子显微镜的应用有利于成像通过厚的体积的vibratome部分。 与自动图像处理软件组合,厚的淋巴结横截面图像还有助于在组织的相对大的区域内的细胞事件的定量,从而提供关于感兴趣的细胞事件的空间分布的更清晰的图像(例如 。,T细胞聚类)。 该方法也可以容易地应用于其它组织,例如脾或皮肤。

关键字:双光子显微镜, 淋巴结, T淋巴细胞, 振动切片机, 免疫反应

材料和试剂

  1. 剃刀刀片,单边,9号(0.22mm)(VWR国际,目录号:55411-050)
  2. Greiner CELLSTAR 24孔平底板(Sigma-Aldrich,目录号:M8812)
  3. 96孔圆底板(Corning,目录号:3788)
  4. Durapore胶带(3M,目录号:1538-1)
  5. Superfrost TM和ColorFrost TM TM载玻片(25×75×1.0mm)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:4951PLUS4 )
  6. Coverslips,No.1.5(ProSciTech,目录号:G425-2460)
  7. 琼脂糖(Sigma-Aldrich,目录号:A6013-250G)
  8. Vetbond组织粘合剂(3M,目录号:1469SB)
  9. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM ,目录号:10010023)
  10. 蛋白质块(Dako,目录号:X0909)
  11. 正常驴血清(NDS)(Jackson ImmunoResearch,目录号:017-000-121)
  12. 长寿黄金抗衰退试剂(Thermo Fisher Scientific,Molecular Probes TM ,目录号:P36930)
  13. 抗体[例如,pacific blue anti-B220,RA3-6B2(Biolegend,目录号:103230); 山羊抗CD69(R& D Systems,目录号:AF2386)]
  14. 高碘酸钠(NaIO 4)(Sigma-Aldrich,目录号:311448)
  15. 正磷酸二氢钠一水合物(NaH 2 PO 4·H 2 O)(一碱价)(VWR,目录号:97062-412) br />
  16. 无水(Na 2 HPO 4)(二价)(Ajax Finechem,目录号:621-500G)的二 - 正磷酸氢钠
  17. L-赖氨酸(Sigma-Aldrich,目录号:L5501)
  18. 来自猪皮的明胶(Sigma-Aldrich,目录号:G1890-100G)
  19. 用于分子生物学的甘油(≥99%)(Sigma-Aldrich,目录号:G5516)
  20. 16%多聚甲醛水溶液(Electron Microscopy Sciences,目录号:30525-89-4)
  21. 高碘酸 - 赖氨酸 - 多聚甲醛(PLP)固定剂(参见配方)
  22. P缓冲区(参见配方)
  23. L-赖氨酸(参见配方)
  24. 5%明胶/甘油混合物(见配方)

设备

  1. Vibratome(Leica,型号:VT1200S)
  2. Dumont No.5镊子(Fine Science Tools,目录号:11251-10)
  3. 剪刀,直,锋利(Roboz,目录号:RS-6752)
  4. 软毛刷漆刷(,例如:2/0)
  5. 具有可调相干变色龙Ti:Sa激光器(ZEISS,型号:LSM 710NLO)的显微镜
    注意:此产品已停产(可更换产品,例如ZEISS,型号:LSM 800)。
  6. 称量1克的秤
  7. 微波

软件

  1. Imaris,Bitplane( http://www.bitplane.com/

程序

  1. 收获淋巴结(LN),并置于2 ml PLP或2%多聚甲醛(PFA)在4°C固定。
    注意:
    1. 该步骤是可选的,但对于保持细胞形态至关重要,但可能损害某些抗体染色。 我们测试的一个实例是抗-XCR1(克隆:ZET),其中固定消除染色。 PLP优选用于保持细胞细胞形态,例如细胞树突。
    2. 涉及动物的所有程序必须根据经过适当培训和认证的人员的认可机构,地区或国家协议(如适用)进行。
  2. 固定后(对于小LN,为4-6小时,对于较大的组织,例如炎症后肿胀的LN,达12小时),将组织转移到2ml冷PBS中并保持在冰上。
  3. 将组织嵌入琼脂糖凝胶中:
    1. 将温热的2%琼脂糖在PBS(煮沸,然后在流动的自来水下冷却,直到温暖接触)倒入24孔板的孔中;
    2. 将组织装入装有温热琼脂糖的孔中并按所需方向,图1(每个LN应接触孔底部,并且随后平行于该平面切片);
    3. 确保平板是平的,留在冰上,让琼脂糖凝固

      图1.将琼脂糖凝胶中的LN包埋。将温热的琼脂糖倒入孔中,将LN包埋在琼脂糖中并调整到所需的方向(左)。允许琼脂糖凝固(右)。

  4. 同时,按照制造商的说明启动振动筛并校准切割刀片。准备冰浴,并用冷PBS填充缓冲液托盘,如图2所示

    图2.设置vibratome 。插入刀片并校准,准备冰浴,并用冷盐水缓冲液填充缓冲盘。

  5. 用一把镊子取出含有LN的琼脂糖块,用刀片小心地修剪成长方体形状,如图3所示。
  6. 将1-2条3M Durapore胶带贴在样品架上。使用一滴Vetbond粘合剂(图3,右)将修剪好的LN /琼脂糖块固定在样品架上(LN在块顶部附近)。
    注:优选由制造商推荐的氰基丙烯酸酯胶。但是,如果使用Vetbond,请等待〜1分钟后粘贴组织块以使胶水干燥。


    图3.安装LN /琼脂糖块。从孔(左)中取出集琼脂糖凝胶,用刀片修剪成长方体形状(中间),并将块安装到样品架上,少量胶水(右)。

  7. 将样品支架安装到vibratome上进行切割。 LN应该对齐,使其纵向剖分,我们发现组织质量更好保存(图4)
  8. 设置vibratome确保适当的切割速度和厚度。通常,我们使用〜0.03 mm/s刀片速度,〜1.6 mm幅度,以及〜200-250μm厚度,以最好地保护LN结构完整性。
  9. 首先在琼脂糖表面上方的振动叶片,使用刷子作为指导,并开始通过自动单一或连续切片切割。丢弃第一个切片。
  10. 将切片(用5号钳和软毛刷)转移到填充有冷PBS的96孔圆底板中的孔中,图4.特别小心只夹住切片的琼脂糖部分,破坏LN组织

    图4.处理vibratome切片。一旦LN /琼脂糖切片被切割(左),使用一对细镊子和刷子从缓冲盘(中间)检索切片,离开在冷PBS中。作为参考,切片应显得相对完整,对组织的破坏最小(右图)
  11. 通过用抗体染色的连续步骤中使用的试剂填充每一行来制备用于抗体染色的96孔圆底板(图5):
    行1:冷PBS(其中LN切片最初放置)
    第2行:蛋白质块
    第3行:第一抗体(〜70μl每孔)
    第4行:PBS(Wash#1)
    第5行:PBS(清洗#2)
    第6行:第二抗体(〜70μl/孔)
    第7行:PBS(Wash#1)
    行8:PBS(清洗#2)
  12. 在每个孵育步骤后,将LN切片转移到每列
  13. 在蛋白质块中孵育1-2小时
  14. 转移到含有一抗(稀释于2.5%NDS)的孔中,并在4℃下孵育8小时或过夜。
    注意:我们测试了太平洋蓝结合的抗B220(稀释1:200)和未结合的抗CD69(稀释1:50)。 应注意,每个抗体批次应单独滴定以获得最佳结果。
  15. 转移到第一个洗涤缓冲液(PBS)孔20-30分钟,然后第二个孔含有洗涤缓冲液(PBS)。
  16. 转移到含有二抗(在2.5%NDS中稀释)的孔中,并在4℃下孵育8小时或过夜。
  17. 转移到第一个洗涤缓冲液(PBS)孔20分钟,然后第二个孔含有洗涤缓冲液(PBS)

    图5.抗体染色的平板设置每个孔含有一个LN切片,其沿每个柱转移以进行抗体标记和洗涤步骤。

  18. 将LN切片放在玻璃显微镜载玻片上(每片至多4个LN切片)。 按所需方向排列。
  19. 使用PBS中的5%明胶/甘油作为安装介质,并仔细地将盖玻片覆盖在组织上,使边缘被密封。
  20. 幻灯片现在可以进行双光子成像:
    采集参数不同,取决于所需的质量和荧光团配置(参见注释)。
    在我们的实验中,我们使用:Zeiss LSM710 NLO与可调相干变色龙Ti:Sa激光蔡司20x/1.0 NA水浸镜头。
    分辨率:512 x 512
    切片间隔:2.5μm
    缩放:〜1.5x
    激光功率和增益设置的深度调整校正,以减少组织的过度激光激发,从而导致光漂白和光毒性。
    使用Zeiss Zen软件的瓦片扫描功能获取整个LN横截面。示例如图6所示。
  21. 可以在专用图像处理软件(例如,Bitplane Imaris)上执行采集后处理,以允许对单元(点特征函数)进行定量和输出统计量。此外,Imaris XTension函数或定制的MATLAB脚本可用于进一步分析。

代表数据



图6.使用双光子显微 scopy获得的LN切片的实例图像示出了由CellTrace紫罗兰标记的CD4T细胞填充的150μm切片的小鼠腹股沟淋巴结LN绿色),CellTracker深红色标记的CD8 T细胞(白色)和TRITC标记的迁移性DC(红色)。根据制造商方案(Thermo Fisher Scienctific)用CellTrace或CellTracker染料标记细胞。然后将LN切片用Pacific Blue抗-B220(蓝色)染色。另外在蓝色是描绘通过二次谐波发生照明的LN胶囊的胶原。使用Zeiss LSM710NLO在双波长(800nm用于激发CellTrace紫,CellTracker深红和太平洋蓝,和880nm用于TRITC激发)和512×512分辨率的8×4瓦片中获得图像,用于3×1.5mm LN横截面。然后使用Imaris(位平面)处理图像。使用Imaris XTension中的通道算术函数去除自发荧光和光谱交叉。

笔记

  1. 在较慢的切割速度下可获得最佳的组织质量。我们发现0.03毫米/秒切片组织与最小的破坏。然而,如果认为可接受,则可以增加切割速度以节省时间
  2. Prolong Gold固定介质也可以与明胶/甘油结合使用,以利用它们的防褪色性能:在组织切片的顶部放置小滴的长期黄金,和明胶/甘油混合在幻灯片的空白区域。这将允许Prolong Gold覆盖组织切片,同时允许明胶/甘油混合物有效地密封载玻片的边缘。
  3. 虽然共聚焦显微镜可以用于获取vibratome切片的图像(我们已达到40微米的深度),它不能成像足够深,以充分利用切片的厚度。双光子 显微镜适用于成像高达150-200微米的深度
  4. 取决于荧光团组合,双重双光子激发波长可以用于获得具有不同激发截面的荧光(例如,对AlexaFluor染料使用800nm,对于EGFP/DsRed使用920nm)。
  5. 某些荧光团被双光子激发快速漂白,应该仔细考虑。 例如,我们发现缀合AlexaFluor 647的抗体是这种目的的不良选择。 相比之下,AlexaFluor 594对于双光子成像是相对光稳定的。

食谱

  1. 高碘酸 - 赖氨酸 - 多聚甲醛固定剂
    对于10ml溶液:
    3.75ml 0.1M P-缓冲液(pH7.4) 3.75ml 0.2M L-赖氨酸(pH7.4) 0.0213g NaIO 4
    2.5ml 4%多聚甲醛
    注意:每次使用时都要新鲜准备。
  2. P-缓冲液,0.1M/mL 对于200ml溶液:
    19ml 0.2M NaH 2 PO 4(一碱价)
    81ml 0.2M Na 2 HPO 4(二价)
    100ml H 2 O 2 / 将pH调节至7.4
    存储在4°C
  3. L-赖氨酸,0.2μM/mL 对于100ml溶液:
    2.92g L-赖氨酸 100ml 0.1M P-缓冲液
    将pH调节至7.4
    存储在4°C
  4. 5%明胶/甘油混合物
    对于20ml溶液:
    1克来自猪皮的明胶
    1 ml甘油用于分子生物学,≥99%
    20ml PBS
    通过倒置管混合并置于37℃水浴中以防止凝胶硬化
    注意: 每次使用时都会重新更新。

致谢

该方案改编自我们的出版物(Hor等人,2015)。 这项工作得到国家卫生和医学研究委员会和澳大利亚研究委员会的支持。

参考文献

  1. Hor,JL,Whitney,PG,Zaid,A.,Brooks,AG,Heath,WRand Mueller,SN(2015)。  与树突状细胞亚群的时空不同的相互作用促进CD4 + 和CD8 + T细胞激活到局部病毒感染 。 免疫 43(3):554-565。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Hor, J. L. and Mueller, S. N. (2016). Imaging Thick Lymph Node Tissue Sections. Bio-protocol 6(18): e1938. DOI: 10.21769/BioProtoc.1938.
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