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Dissection and Staining of Mouse Brain Ventricular Wall for the Analysis of Ependymal Cell Cilia Organization
小鼠脑室管膜细胞纤毛组织分析:脑室的解剖和染色   

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Abstract

In the developing and mature central nervous system (CNS) the ventricular lumen is lined by the neuroepithelium and ependymal, respectively. These ventricular epithelia perform important functions related to the development, morphogenesis and physiology of the brain. In the mature CNS, ependyma constitutes a barrier between brain parenchyma and cerebro- spinal fluid (CSF). The most prominent feature of the apical surface of ependymal cells is the presence of multiple motile cilia that extend towards the ventricular lumen. The beating of cilia ensures the circulation of the CSF and its impairment leads to hydrocephalus. For an effective CSF flow, ciliary beating must be coordinated at the level of individual cells and at the tissue level. This coordination is achieved through the precise organization of cilia positioning within the plane of the ependyma. Two major features have been described regarding the planar organization of cilia in ependymal cells (Mirzadeh et al., 2010) and both have a cellular and tissular aspect (Boutin et al., 2014). The first one, rotational polarity, refers to the orientation of ciliary beating. At the cellular level, all cilia beat in the same direction (Figure 1B, black arrows). At the tissue level, each ependymal cell coordinates the direction of their beating with that of neighboring cells (Figure 1C, grey arrows). The second feature, translational polarity, is unique to ependymal cells and refers to the clustering of cilia in a tuft. At the cellular level, this tuft is displaced relative to the center of the ependymal cell (Figure 1B, red arrow). At the tissue level, the positioning of the ciliary tuft is coordinated between adjacent cells (Figure 1C). Alteration of any of these polarities at either level impairs CSF flow circulation (Mirzadeh et al., 2010; Boutin et al., 2014; Guirao et al., 2010; Hirota et al., 2010; Ohata et al., 2014). Cilia axonemes arise from basal bodies (BB) which are cylindrical structures anchored perpendicular to the sub-apical surface of the cells (Figure 1D). BBs are polarized by the presence of appendices such as basal foot or striated rootlets. The basal foot protrudes in a direction correlated with the direction of cilia beating, while the striated rootlet protrudes in the opposite direction of cilia beating (Marshall, 2008). The ‘en face view’ observation of BBs’ organization allows the visualization of ependymal polarities (Mirzadeh et al., 2010; Boutin et al., 2014). Here, we describe an immunofluorescence (IF) protocol for observation of ciliated cells in mouse brain ventricular lateral wall whole mounts (LWWM). This protocol can be used for classical confocal microscopy analysis. In addition, it is well suited for super-resolution STimulated Emission Depletion (STED) microscopy if observation of structures that have features which are smaller than the optical diffraction limit is needed. Finally, we describe a combination of antibodies that allow the concomitant observation, in a single sample, of ependymal polarities at the level of individual cilia, individual cells and at the tissue level.

Materials and Reagents

  1. Microscope slide
  2. Coverslips
  3. Mice at desired stage [between postnatal day 1 (P1) and 21 (P21)]
  4. 1x PBS 
  5. Triton X-100
  6. 4% Paraformaldehyde (PFA) (prepared by dilution of powder in 1x PBS)
  7. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A-8022 )
  8. Hoechst 33258 solution (Sigma-Aldrich, catalog number: 94403 )
  9. Mowiol (Merck Millipore Corporation, Calbiochem®, catalog number: 475904 )
  10. Prolong Gold antifade reagent (Thermo Fisher Scientific, Molecular ProbeTM, catalog number: P36934 )
  11. ZO1 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 61-7300 )
  12. γ-tubulin [GTU-88] (Abcam, catalog number: ab11316 )
  13. FGFR1OP (FOP) (Abnova Corporation, catalog number: H00011116-M01 )
  14. β-catenin (BD, catalog number: 610153 )
  15. FoxJ1 (eBioscience, catalog number: 14-9965 )
  16. Acetylated-α-tubulin (Sigma-Aldrich, catalog number: T6793 )
  17. Secondary antibodies
    1. Anti-Rabbit A647 (Invitrogen, catalog number: A21244 )
    2. Anti-Mouse IgG1 A568 (Invitrogen, catalog number: A21124 )
    3. Anti-Mouse IgG2b A488 (Invitrogen, catalog number: A21141 )
    4. Anti-Mouse IgG1 A488 (Invitrogen, catalog number: A21121 )
    5. Anti-Mouse IgG2b A568 (Invitrogen, catalog number: A21144 )
  18. Primary and secondary antibodies (see Recipes)

Equipment

  1. Dissection tools including forceps (Zillow, Dumont, model: 55 ) and Ultra Fine Micro Knives (Fine Science Tools, catalog number: 10316-14 )
  2. Confocal microscope (OLYMPUS, model: Fluoview FV1000 )
  3. STED microscope (Leica Microsystems, model: SP8 3x STED ) equipped with a 100x oil objective (HC-PL-APO; NA 1.40; STED white) and a 592 nm depletion laser

Software

  1. Biotool software (Boutin et al., 2014)

Procedure

The general procedure described here is suitable for staining ependymal cells from lateral wall whole mounts of P1 to P21 mice. However, the last sub-dissection step of the tissue has to be performed before the staining for mice older than P5 and after the staining for mice younger than P5 to ensure an optimal result.

  1. After cervical dislocation of the mice, the brain is removed from the skull.
  2. Dissection of the brain at RT in 1x PBS to reveal the ventricular lateral wall (Figure 2) [a protocol including a video is provided in Mirzadeh et al. (2010)].
  3. Fixation of the whole mount by immersion in 500 μl fresh solution of 4% PFA-0.1% Triton X-100 12 min at room temperature (RT).

  4. Wash 3 times 10 min in 1x PBS-0.1% Triton X-100 (1 ml) at RT with agitation.

  5. Sub-dissection of the lateral wall by separating it from the underlying striatum [see Mirzadeh et al. (2010) for a precise description of this step]. Performing this sub-dissection at this step of the protocol improves the staining for tissues older than P5.
  6. Incubation in blocking solution 1x PBS-3% BSA (1 ml) 1 h RT with agitation.

  7. Incubation with primary antibody diluted in 1x PBS-3% BSA (250-500 μl) over-night RT with agitation.

  8. Wash 3 times 1x PBS -0.1% Triton X-100 (1 ml) 10 min RT with agitation.

  9. Incubate with secondary antibody (1:800) and Hoechst (1:1,000) diluted in 1x PBS (For confocal observation of samples) OR with secondary antibody (1:800) diluted in 1x PBS only (for STED observation of samples) (800 μl) 1 h RT with agitation.

  10. Wash 3 times 1x PBS-0.1% Triton X-100 (1 ml) 10 min RT with agitation.

  11. For tissues younger than P5: sub-dissection of the LWWM by separating the lateral wall and the underlying striatum in 1x PBS [see Mirzadeh et al. (2010)].
  12. Place the whole mount on the slide with ependymal face up.

  13. Add 7-8 drops of Mowiol (for confocal observation of samples) or of Prolong mounting medium (for STED observation of samples) directly on the whole mount.
  14. Place a coverslip on the sample.
  15. Keep slides at room temperature, protected from the light, for at least 24 h before imaging.

Representative data


Figure 1. Planar organization of ependymal cells in the lateral wall. A. Schematic representation of the mouse forebrain depicting the localization of the lateral wall of the lateral ventricle. Dashed square highlights the region of interest considered for analysis of ependymal planar polarity. B-C. Schemes represent the planar organization of ependymal cells in the region of interest. B. At the cellular level, ependymal cilia are clustered in a tuft (dotted red line) which localize off center of the apical surface (red arrow). Ciliary beatings (small black arrows) are coordinated. The sum of individual ciliary beatings generates the tuft beating direction (Thick grey arrow). In individual cells, the direction of tuft positioning correlates with the direction of tuft beating (C). At the tissue level, beating direction (grey arrows) and positioning of the ciliary tuft (red arrows) are coordinated between neighboring ependymal cells. D-E. Schematic representation of the lateral (D) and ‘en face’ (E) view of individual ependymal cell organization. D. Multicilia axonemes arise from basal bodies (BBs) anchored perpendicularly to the apical surface of the cells (green). BBs display polarized appendices such as the basal foot (red dot) that indicates the effective beating direction of the cilium (small black arrow). E. Observation of cilia BBs from a top view reveals all ependymal polarity features.


Figure 2. Dissection steps for LWWM preparation. LWWM preparations allows the “en face” observation of ependymal cells. A. Brain after removal from the skull. B. Cut the brain between the two hemispheres (dashed red line) using a microscalpel. C. Remove the cerebellum (dashed red line). D-F. Use a microscapel to reveal the hippocampus (dashed red line). G-H. Insert the microscalpel in the ventricle (arrow) to remove the hippocampus and medial ventricular wall. I. Remove cortical wall following the dashed red line. J. Whole mount ready to be fixed, the lateral wall is outlined by the dashed red line.


Figure 3. Examples of staining on P1, P4, P5 and P21 LWWM Confocal (A-E) and STED (F) images. LWWM stained for A. ZO1 (green) and Acetyl-α- tubulin (red) at P1. B. FoxJ1 (green) and DAPI (blue) at P4. C. β-catenin (green) and Acetyl-α- tubulin (red) at P21. D. β-catenin (green) and ɣ-tubulin (red) at P21. E-F. FGFR1 Oncogene Partner (FOP) at P5. Scale bar: 10 μm in A-B, 15 μm in C-D and 2.5 μm in E-F.


Figure 4. Analysis of planar organization of ependymal cells in the lateral wall (A, B, C). Triple immunostaining for ZO1 (white), FGFR1 Oncogene Partner (FOP, green) and γ-tubulin (red) on P21 LWWM allows the observation of translational and rotational polarities at the cellular and tissular level in a single sample. A. Large field picture displaying the tissue organization. B. Zoom on the cell marked with * in (A). C. Zoom on the BBs patch from cell shown in (B). Analysis of ZO1/FOP/γ-tubulin triple staining using Biotool1 software [described in Boutin et al. (2014)] allows the definition of polarity parameters at BB, cell and tissue level. C’. Definition of individual cilia polarity: each black vector represents the rotational polarity axis of individual BB defined as the direction from FOP (green) to γ-tubulin (red) positive dots. The biotool software calculates the mean BB orientation of the patch to define the cellular rotational polarity axis that corresponds to the beating direction of individual cell (grey arrow). B’-B”. Definition of polarity axis at the cellular level: Outline of cell (green) and BB patch (red). The grey vector in (B’) represents the rotational polarity axis of the cell defined as shown in C’. The red arrow in (B’’) represents the translational polarity axis of the cell defined as direction from the cell center (green cross) to the BB patch center (red cross). A’-A”. Analysis of polarity at the tissue level: The translational and rotational polarity axes are defined for each cell and compared to mean direction of the field (black and red thick arrows). Scale bar: 15 μm in A, 2.5 μm in B and 1 μm in C.

Notes

  1. Freshly made 4% PFA aliquots can be stored at -20 °C up to one year and defrosted just before fixation.
  2. Short fixation time is mandatory for good staining. 12 min fixation is sufficient to fix large and small tissues while preserving the structure and giving the best results. We recommend not to exceed 20 min fixation for ependymal observation.

Recipes

  1. Primary antibodies with the corresponding secondary antibodies

    Table 1. List of primary and secondary antibodies
    Antibody
    Isotype
    Reference
    Dilution
    Secondary antibody
    ZO1
    Rabbit
    Invitrogen 61-7300
    1:600
    Anti-Rabbit A647
    γ-tubulin [GTU-88]
    Mouse IgG1
    Abcam ab11316

    1:400
    Anti-Mouse IgG1 A568
    FGFR1OP (FOP)
    Mouse IgG2b
    Abnova H00011116-M01
    1:2,000
    Anti-Mouse IgG2b A488
    β-catenin
    Mouse IgG1
    BD Transduction Laboratories 610153
    1:1,000
    Anti-Mouse IgG1 A488
    FoxJ1
    Mouse IgG1
    eBioscience 14-9965

    1:2,000
    Anti-Mouse IgG1 A488
    Acetylated-α- tubulin
    Mouse IgG2b
    Sigma T6793
    1:1,000
    Anti-Mouse IgG2b A568

Acknowledgments

The method described in this article has been optimized for detection of ependymal cells organization and is based on a protocol described previously (Mirzadeh et al., 2010). The original version of the protocol has been described in (Boutin et al., 2014). The authors wish to thank the Spassky lab (ENS, Paris) for introduction to the LWWM dissection method. CB is a recipient of a postdoctoral fellowship from ‘La Ligue Nationale Contre le Cancer’. HC acknowledges funding from the Agence Nationale de la Recherche (ANR-13-BSV4-0013-01-ATMIR). This work was supported by the French National Research Agency through the "Investments for the Future" program (France-BioImaging, ANR-10-INSB-04).

References

  1. Boutin, C., Labedan, P., Dimidschstein, J., Richard, F., Cremer, H., Andre, P., Yang, Y., Montcouquiol, M., Goffinet, A. M. and Tissir, F. (2014). A dual role for planar cell polarity genes in ciliated cells. Proc Natl Acad Sci U S A 111(30): E3129-3138.
  2. Guirao, B., Meunier, A., Mortaud, S., Aguilar, A., Corsi, J. M., Strehl, L., Hirota, Y., Desoeuvre, A., Boutin, C., Han, Y. G., Mirzadeh, Z., Cremer, H., Montcouquiol, M., Sawamoto, K. and Spassky, N. (2010). Coupling between hydrodynamic forces and planar cell polarity orients mammalian motile cilia. Nature Cell Biology 12(4): 341-U386.
  3. Hirota, Y., Meunier, A., Huang, S., Shimozawa, T., Yamada, O., Kida, Y. S., Inoue, M., Ito, T., Kato, H., Sakaguchi, M., Sunabori, T., Nakaya, M. A., Nonaka, S., Ogura, T., Higuchi, H., Okano, H., Spassky, N. and Sawamoto, K. (2010). Planar polarity of multiciliated ependymal cells involves the anterior migration of basal bodies regulated by non-muscle myosin II. Development 137(18): 3037-3046.
  4. Marshall, W. F. (2008). Basal bodies platforms for building cilia. Curr Top Dev Biol 85: 1-22.
  5. Mirzadeh, Z., Doetsch, F., Sawamoto, K., Wichterle, H. and Alvarez-Buylla, A. (2010). The subventricular zone en-face: wholemount staining and ependymal flow. J Vis Exp(39).
  6. Mirzadeh, Z., Han, Y. G., Soriano-Navarro, M., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. (2010). Cilia organize ependymal planar polarity. J Neurosci 30(7): 2600-2610.
  7. Ohata, S., Nakatani, J., Herranz-Perez, V., Cheng, J., Belinson, H., Inubushi, T., Snider, W. D., Garcia-Verdugo, J. M., Wynshaw-Boris, A. and Alvarez-Buylla, A. (2014). Loss of Dishevelleds disrupts planar polarity in ependymal motile cilia and results in hydrocephalus. Neuron 83(3): 558-571.

简介

在发展中和成熟的中枢神经系统(CNS)中,心室腔分别由神经上皮和室管膜排列。这些心室上皮执行与脑的发育,形态发生和生理相关的重要功能。在成熟CNS中,室管膜构成脑实质和脑脊液(CSF)之间的屏障。室管膜细胞的顶面的最突出的特征是存在向心室腔延伸的多个运动性纤毛。纤毛的跳动确保CSF的循环,并且其损伤导致脑积水。对于有效的CSF流动,睫状细胞跳动必须在单个细胞水平和组织水平协调。这种协调是通过精确组织在室内平面内的纤毛定位来实现的。已经描述了关于室管膜细胞中纤毛的平面组织的两个主要特征(Mirzadeh等人,2010),并且它们都具有细胞和组织方面(Boutin等人 ,2014)。第一个,旋转极性,指睫毛跳动的方向。在细胞水平,所有纤毛在相同方向跳动(图1B,黑色箭头)。在组织水平,每个室管膜细胞协调其拍打的方向与相邻细胞的方向(图1C,灰色箭头)。第二个特征,翻译极性,对室管膜细胞是唯一的,是指簇中的纤毛聚集。在细胞水平,该簇相对于室管膜细胞的中心位移(图1B,红色箭头)。在组织水平,睫状簇的定位在相邻细胞之间协调(图1C)。在任一水平上改变任何这些极性都会损害CSF流动循环(Mirzadeh等人,2010; Boutin等人,2014; Guirao等人,/em,2010; Hirota等人,2010; Ohata等人,2014)。纤毛轴突起源于基底体(BB),其是垂直于细胞的子根表面锚定的圆柱形结构(图1D)。 BB通过附属物例如基底足或条纹小根的存在而极化。基底足在与纤毛搏动方向相关的方向上突出,而条纹根在纤毛搏动的相反方向突出(Marshall,2008)。 BBs组织的"正面观察"观察允许室管膜极性的可视化(Mirzadeh等人,2010; Boutin等人,2014)。在这里,我们描述免疫荧光(IF)协议观察的纤毛细胞对小鼠脑室侧壁整体(LWWM)。此协议可用于经典共聚焦显微镜分析。此外,如果需要观察具有小于光学衍射极限的特征的结构,它非常适合于超分辨率刺激发射损耗(STED)显微术。最后,我们描述了允许在单个样品中伴随观察在个体纤毛,单个细胞和组织水平的室管膜极性的抗体的组合。

材料和试剂

  1. 显微镜载玻片
  2. 盖舌
  3. 在期望阶段(在出生后第1天(P1)和21(P21)之间)的小鼠
  4. 1x PBS 
  5. Triton X-100
  6. 4%多聚甲醛(PFA)(通过在1x PBS中稀释粉末制备)
  7. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A-8022)
  8. Hoechst 33258溶液(Sigma-Aldrich,目录号:94403)
  9. Mowiol(Merck Millipore Corporation,Calbiochem ,目录号:475904)
  10. Prolong Gold抗衰减试剂(Thermo Fisher Scientific,Molecular Probe TM ,目录号:P36934)
  11. ZO1(Thermo Fisher Scientific,Invitrogen TM,目录号:61-7300)
  12. γ-微管蛋白[GTU-88](Abcam,目录号:ab11316)
  13. FGFR1OP(FOP)(Abnova Corporation,目录号:H00011116-M01)
  14. β-连环蛋白(BD,目录号:610153)
  15. FoxJ1(eBioscience,目录号:14-9965)
  16. 乙酰化-α-微管蛋白(Sigma-Aldrich,目录号:T6793)
  17. 二抗
    1. 抗兔A647(Invitrogen,目录号:A21244)
    2. 抗小鼠IgG1A568(Invitrogen,目录号:A21124)
    3. 抗小鼠IgG2b A488(Invitrogen,目录号:A21141)
    4. 抗小鼠IgG1 A488(Invitrogen,目录号:A21121)
    5. 抗小鼠IgG2b A568(Invitrogen,目录号:A21144)
  18. 一抗和二抗(见配方)

设备

  1. 解剖工具包括镊子(Zillow,Dumont,型号:55)和超细微刀(Fine Science Tools,目录号:10316-14)
  2. 共聚焦显微镜(OLYMPUS,型号:Fluoview FV1000)
  3. 装备有100×油物镜(HC-PL-APO; NA 1.40; STED白)和592nm耗尽激光器的STED显微镜(Leica Microsystems,型号:SP8 3x STED)

软件

  1. Biotool软件(Boutin等人,2014)

程序

这里描述的一般程序在此适合于描述适合于从P1到P21小鼠的侧壁整个支架染色室管膜细胞。然而,组织的最后一个亚解剖步骤必须在P5以前的小鼠的染色之前和P5以下的小鼠的染色之后进行,以确保最佳结果。

  1. 在小鼠颈椎脱位后,从颅骨中取出脑。
  2. 在室温下在1x PBS中解剖大脑以显示心室 侧壁(图2)[包括视频的协议被提供 Mirzadeh et al。 (2010)]。
  3. 通过浸没固定整个安装 ?在500μl新鲜的4%PFA-0.1%Triton X-100溶液中12分钟 温度(RT)。
  4. 在室温下,在搅拌下,在1x PBS-0.1%Triton X-100(1ml)中洗涤3次10分钟。
  5. 对于P5以下的组织:LWWM的解剖 在1x PBS中分离侧壁和下面的纹状体[参见 Mirzadeh et al。(2010)]。
  6. 将整个安装在幻灯片上,室内表面朝上。
  7. 添加7-8滴Mowiol(用于样品的共焦观察)或 ?长期安装介质(用于样品的STED观察)直接 整个座架。
  8. 将盖玻片放在样品上。
  9. 保持幻灯片在室温,保护免受光,至少24小时成像。

代表数据


图1。 侧壁中室管膜细胞的平面组织 A.小鼠前脑的示意图,描绘侧脑室侧壁的定位。虚线方框突出显示了考虑用于分析室管膜平面极性的感兴趣区域。公元前。方案代表感兴趣区域中室管膜细胞的平面组织。 B.在细胞水平,室管膜纤毛聚集在一簇(红色虚线),其定位在顶端表面的中心(红色箭头)。睫状鞭毛(小黑箭头)是协调的。单个睫毛打击的总和产生簇打打方向(粗灰色箭头)。在单个细胞中,绒头定位的方向与簇绒方向(C)相关。在组织水平,搏动方向(灰色箭头)和睫状体束(红色箭头)的定位在相邻的室管膜细胞之间协调。 D-E。个体室管膜细胞组织的横向(D)和"en面"(E)视图的示意图。 D.多纤维素轴突产生于垂直锚定于细胞顶端表面的基底体(BB)(绿色)。 BB显示极化附录,例如指示纤毛的有效搏动方向(小黑色箭头)的基底脚(红点)。 E.从顶视图观察纤毛BB显示所有的室内极性特征

图2. LWWM准备的解剖步骤。 LWWM制剂允许"en面"观察室管膜细胞。 A.从头骨中取出后的脑。 B.使用微型手术切割两个半球之间的大脑(红色虚线)。 C.取出小脑(红色虚线)。 D-F。使用micropapel显示海马(红色虚线)。 G-H。插入微脉冲在心室(箭头),以去除海马和内侧心室壁。 I.沿着红色虚线除去皮质壁。 J.整个安装准备固定,侧壁由红色虚线勾勒出来。


图3.在P1,P4,P5和P21LWWM共聚焦(A-E)和STED(F)图像上染色的实施例。 LWWM在P1处染色A.ZO1(绿色)和乙酰基-α-微管蛋白(红色)。 B.PoxJ1(绿色)和DAPI(蓝色)。 C.在P??21的β-连环蛋白(绿色)和乙酰基-α-微管蛋白(红色)。 D.β-catenin(绿色)和P - 微管蛋白(红色)。 E-F。 FGFR1癌基因合作伙伴(FOP)。比例尺:A-B为10μm,C-D为15μm,E-F为2.5μm

图4.侧壁中室管膜细胞的平面组织分析(A,B,C)。在P21 LWWM上的ZO1(白色),FGFR1癌基因合作伙伴(FOP,绿色)和γ-微管蛋白(红色)的三重免疫染色允许在单个样品中观察细胞和组织水平的翻译和旋转极性。 A.显示组织组织的大场图片。 B.放大(A)中标有*的单元格。 C.放大BBs补丁从细胞展示在(B)。使用Biotool1软件(描述于Boutin等人(2014)]的ZO1/FOP /β微管蛋白三重染色的分析允许在BB,细胞和组织水平上定义极性参数。 C'。个体纤毛极性的定义:每个黑色矢量代表单个BB的旋转极性轴,其定义为从FOP(绿色)到μ-微管蛋白(红色)正点的方向。生物工具软件计算贴片的平均BB取向,以限定与单个细胞(灰色箭头)的拍打方向对应的细胞旋转极性轴。 B'-B"。细胞水平极性轴的定义:细胞(绿色)和BB补丁(红色)的轮廓。 (B')中的灰色向量表示如C'所示定义的单元的旋转极性轴。 (B")中的红色箭头表示被定义为从细胞中心(绿色交叉)到BB斑块中心(红色叉)的方向的细胞的平移极性轴。 A'-A"。组织水平极性分析:为每个细胞定义平移和旋转极性轴,并与场的平均方向(黑色和红色粗箭头)进行比较。比例尺:A为15μm,B为2.5μm,C为1μm。

笔记

  1. 新鲜制备的4%PFA等分试样可以在-20℃下储存至多一年,并在固定之前解冻。
  2. 短的固色时间是良好染色的强制性。 12分钟的固定足以固定大和小的组织,同时保留结构并给出最好的结果。我们建议不要超过20分钟固定室管膜观察。

食谱

  1. 具有相应二级抗体的一级抗体

    表1.主要和次要抗体列表
    抗体
    同种型
    参考
    稀释
    二抗
    ZO1
    兔子
    Invitrogen 61-7300
    1:600
    抗兔A647
    γ-微管蛋白[GTU-88]
    小鼠IgG1
    abcam ab11316

    1:400
    抗小鼠IgG1 A568
    FGFR1OP(FOP)
    小鼠IgG2b
    Abnova H00011116-M01
    1:2,000
    抗小鼠IgG2b A488
    β-连环蛋白 小鼠IgG1
    BD Transduction Laboratories 610153
    1:1,000
    抗小鼠IgG1 A488
    FoxJ1
    小鼠IgG1
    eBioscience 14-9965

    1:2,000
    抗小鼠IgG1 A488
    乙酰化α-微管蛋白 小鼠IgG2b
    Sigma T6793
    1:1,000
    抗小鼠IgG2b A568

致谢

本文中描述的方法已经优化用于室管膜细胞组织的检测,并且基于先前描述的方案(Mirzadeh等人,2010)。协议的原始版本已在(Boutin等人 ,2014年)中描述。作者希望感谢Spassky实验室(ENS,巴黎)介绍LWWM解剖方法。 CB是"La Ligue Nationale Contrele Cancer"博士后研究员的接受者。 HC承认来自国家民用航空局(ANR-13-BSV4-0013-01-ATMIR)的资金。这项工作得到了法国国家研究机构通过"未来投资"计划(法国 - 生物图像,ANR-10-INSB-04)的支持。

参考文献

  1. Boutin,C.,Labedan,P.,Dimidschstein,J.,Richard,F.,Cremer,H.,Andre,P.,Yang,Y.,Montcouquiol,M.,Goffinet,AM和Tissir, )。 平面细胞极性基因在纤毛细胞中的双重作用。 Proc Natl Acad Sci USA 111(30):E3129-3138。
  2. Guirao,B.,Meunier,A.,Mortaud,S.,Aguilar,A.,Corsi,JM,Strehl,L.,Hirota,Y.,Desoeuvre,A.,Boutin,C.,Han,YG,Mirzadeh, Z.,Cremer,H.,Montcouquiol,M.,Sawamoto,K。和Spassky,N。(2010)。 流体动力和平面细胞极性之间的耦合定位哺乳动物运动性纤毛。自然细胞生物学em> 12(4):341-U386。
  3. Hirota,Y.,Meunier,A.,Huang,S.,Shimozawa,T.,Yamada,O.,Kida,YS,Inoue,M.,Ito,T.,Kato,H.,Sakaguchi,M.,Sunabori ,Nakaya,MA,Nonaka,S.,Ogura,T.,Higuchi,H.,Okano,H.,Spassky,N.and Sawamoto,K。(2010)。 多层室管膜细胞的平面极性涉及由非肌肉肌球蛋白II调节的基底体的前迁移。 开发 137(18):3037-3046
  4. Marshall,W. F.(2008)。 建立纤毛的基础体平台 Curr Top Dev Biol 85:1-22。
  5. Mirzadeh,Z.,Doetsch,F.,Sawamoto,K.,Wichterle,H。和Alvarez-Buylla,A。(2010)。 室下区表面:全身染色和室管膜流动。 Vis Exp (39)。
  6. Mirzadeh,Z.,Han,Y.G.,Soriano-Navarro,M.,Garcia-Verdugo,J.M.and Alvarez-Buylla,A。(2010)。 纤毛组织室内平面极性 J Neurosci 30( 7):2600-2610。
  7. Ohata,S.,Nakatani,J.,Herranz-Perez,V.,Cheng,J.,Belinson,H.,Inubushi,T.,Snider,WD,Garcia-Verdugo,JM,Wynshaw-Boris,A.and Alvarez -Buylla,A。(2014)。 失去失聪会破坏室管膜运动性纤毛的平面极性,并导致脑积水。 Neuron 83(3):558-571。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Labedan, P., Matthews, C., Kodjabachian, L., Cremer, H., Tissir, F. and Boutin, C. (2016). Dissection and Staining of Mouse Brain Ventricular Wall for the Analysis of Ependymal Cell Cilia Organization. Bio-protocol 6(6): e1757. DOI: 10.21769/BioProtoc.1757.
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