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Feb 2019

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Organotypic Slice Culture of the Embryonic Mouse Brain
胚胎小鼠大脑的器官型切片培养   

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Abstract

Organotypic slice culture is a powerful technique for exploring the embryonic development of the mammalian brain. In this protocol we describe a basic slice culture technique we have used for two sets of experiments: axon guidance transplant assays and bead culture assays.

Keywords: Organotypic (器官型), Slice culture (切片培养), Transplant (移植), Axon guidance (轴突导向), Morphogen (成形素), Bead culture (珠子培养)

Background

Organotypic slice culture is a technique that has been widely used in recent years and has been a particularly popular technique in the field of neurodevelopment. It has the great advantage of allowing the culture of developing neural cells in-vitro while maintaining the in-vivo structure of the tissue. In this protocol we describe the slice culture technique we used for two experiments from our recent paper (Clegg et al., 2019). Firstly we performed an axon guidance transplant assay whereby fluorescently labeled tissue was transplanted into a non-fluorescent host slice in order to observe axon outgrowth. This is a modified version of a protocol previously used to study the development of the corpus callosum, but could be easily adapted to examine other axon tracts such as the thalamocortical tract (Niquille et al., 2009). Secondly, we performed a bead culture assay in which beads soaked in recombinant FGF protein was embedded in the tissue, this allowed the examination of the molecular and cellular response of the tissue to the FGF protein. The use of protein soaked beads in this way provides a method for focal delivery of recombinant protein at a specific position, in contrast to bath application which exposes all tissue to the protein equally. This mimics the situation in-vivo where a morphogen (such as FGF17) is expressed at a particular anatomical position and then diffuses through the tissue. This technique could be used to explore the response to a number of different recombinant proteins or pharmacological agents.

Materials and Reagents

Materials

  1. 7 ml bijou tubes (Greiner, catalog number: 189170 )
  2. Pipette tips (Greiner, catalog number: 739288 )
  3. 1.5 ml microcentrifuge tubes (Greiner, catalog number: 616201 )
  4. Peel-A-Way® Embedding Molds (Square–S22, Polysciences)
  5. Falcon® 60 mm TC-treated Center Well Organ Culture Dish (Corning, catalog number: 353037 )
  6. 13 mm Nuclepore polycarbonate track-etched membranes (Whatman, catalog number: 110401 )
  7. Specimen disk
  8. Adhesive

Animals
Mice used in our original study ubiquitously express τGFP and have been described previously (Pratt et al., 2000). All mice were maintained on a CD-1 background (Charles River, strain code: 022). Mice used for timed matings were aged between 6 and 24 weeks.



Reagents
  1. Earls balanced salt solution (EBSS) calcium, magnesium, phenol red (Thermo Fisher, catalog number: 24010043 )
  2. SeaPlaque GTG Agarose (Lonza, catalog number: 50110 )
  3. Affi-Gel Blue gel beads (Bio-Rad, catalog number: 1537301 )
  4. MEM (Thermo Fisher, catalog number: 11090081 )
  5. Neurobasal medium (Thermo Fisher, catalog number: 21103049 )
  6. 1 M HEPES (Thermo Fisher, catalog number: 15630080 )
  7. Penicillin-streptomycin (Thermo Fisher, catalog number: 15140122 )
  8. D-Glucose solution (Merck, catalog number: G8769 )
  9. Gentamicin 50 mg/ml (Thermo Fisher, catalog number: 15750060 )
  10. Glutamine 200 mM (Thermo Fisher, catalog number: 25030081 )
  11. Fetal bovine Serum (Merck, catalog number: F4135 )
  12. B-27 supplement (Thermo Fisher, catalog number: 17504044 )
  13. OCT embedding matrix (Cellpath, catalog number: KMA-0100-00A )
  14. 1x Krebs buffer (from 10x stock, see recipes)
  15. 1x PBS (Thermo Fisher catalog number: 14190094 )
  16. NaCl (Thermo Fisher catalog number: 10428420 )
  17. KCl (Thermo Fisher catalog number: 10684732 )
  18. NaH2PO4 (Thermo Fisher catalog number: 10723621 )
  19. CaCl2 (Thermo Fisher catalog number: 10657662 )
  20. MgCl2 (Thermo Fisher catalog number: 10386743 )
  21. Triton X-100 (Merck, catalog number: 10789704001 )
  22. Bovine serum albumin (Merck, catalog number: A1933 )
  23. Anti-GFP antibody (Abcam, catalog number: ab290 )
  24. Goat anti-Rabbit IgG 488 (Thermo Fisher, catalog number: A-11008 )
  25. DAPI (Thermo Fisher, catalog number: D1306 )
  26. Vectashield Hardset (VectorLabs, catalog number: H-1400 )
  27. 10x Krebs Buffer (see Recipes)
  28. 1x Krebs with antibiotic (see Recipes)
  29. MEM with serum (see Recipes)
  30. Neurobasal (see Recipes)

Equipment

  1. Pipettes
  2. Laminar flow tissue culture hood
  3. Tissue culture incubator (37 °C, 5% CO2)
  4. Dissecting microscope (Euromex, catalog number: DZ1100 )
  5. Leica VT1000 S vibratome
  6. Water bath
  7. Fisherbrand microspatula with flat-ended blade (Thermo Fisher, catalog number: 21-401-20 )
  8. Forceps, size 5 (Fine Science Tools, catalog number: 11251-20 )
  9. Microscissors, 5 mm cutting edge (Fine Science Tools, catalog number: 15003-08 )
  10. Scissors, 9 cm (Fine Science Tools, catalog number: 14060-09 )
  11. 45° Sterile blade (Altomed, model: A10136 )
  12. Cryostat (Leica, catalog number: CM3050 S )
  13. Super-frost Plus slides (Thermo Fisher, catalog number: J1800AMNZ )
  14. Hydrophobic barrier pen (VectorLabs, catalog number: H-4000 )

Procedure

  1. Organotypic slice culture
    1. Oxygenate EBSS by bubbling 100% oxygen through the solution until a color change (pink to orange) is observed. EBSS should then be placed on ice to cool.
    2. Cull pregnant dams at the desired embryonic stage by dislocation of the neck or an overdose of anesthetic.
      Note: Slice cultures can be performed from embryonic day (E) 13.5 onwards. For axon guidance assays a stage should be selected during which axon outgrowth of the tract under investigation is occurring, e.g., E17.5 for the corpus callosum.
    3. Remove the uterus and place in a dish of pre-cooled oxygenated EBSS on ice, in the tissue culture hood, use scissors and forceps to remove embryos from the uterus. Using scissors, cut the head of the embryo from the body, and transfer to the second dish of EBSS.
    4. Use forceps to peel away the skin from the head to reveal the skull, cut down the midline of the skull using microscissors and peel back the skull to reveal the brain. Carefully detach the brain from the base of the head using forceps.
      Note: Brains should be dissected out and placed in EBSS within 1 h of the pregnant dam being culled.
    5. Collect brains in 7 ml bijou tubes containing oxygenated EBSS and place tubes on ice.
      Note: Brains can be held at this stage for up to 3 h to allow time for genotyping (if necessary). It is recommended to add glucose to the EBSS (concentration 0.1%) if planning to hold brains at this stage.
    6. Embed brains individually in approximately 5 ml of low melting point agarose in square embedding molds. Agarose should be held at a temperature of 42 °C using a water bath prior to use. Place mold on ice once the tissue is positioned (Figure 1A).
      Note: Before positioning the brains at the desired plane, stir the agarose surrounding the tissue repeatedly in order to remove any carried over EBSS from the tissue. This prevents tissue separating from the agarose during sectioning (Figure 1A).
    7. Once set, remove agarose from mold and trim using a razor blade until the agarose block containing the tissue is < 1 cm3 (Figure 1B). Attach the agarose block to the specimen disk using adhesive (Figure 1C).
      Note. The plane of section should be determined by the brain region or axon tract under investigation, for corpus callosum axon guidance assays a coronal sectioning plane was used and tissue sections containing the telencephalon were collected.
    8. Section tissue on the vibratome at a section thickness of 400 µm in ice-cold 1x Krebs buffer.
      Note: While sectioning, the vibratome should be set to a frequency setting of 8 and a speed setting of 4. If tissue starts to break up reduce speed to 3.
    9. Collect tissue sections in 1x Krebs containing 100 mM HEPES, 0.5 mg/ml gentamicin and 1% penicillin-streptomycin.
      Note: Transfer of tissue sections should always be done with blunt-ended microspatulas, brushes should not be used as tissue can stick to bristles causing damage.
    10. Add 1 ml MEM with serum to the well of the organ culture dish. Place a Whatman nuclepore membrane onto the MEM at the center of the well (shiny side facing the liquid).
    11. Carefully transfer a single tissue section to the center of the membrane making sure not to submerge any part of the membrane (Figures 1D and 1E). Incubate the culture for 1 h at 37 °C in 5% CO2.
    12. Aspirate MEM and replace with 1 ml Neurobasal medium containing 2% B27 supplement, 1% penicillin-streptomycin, 0.5% glucose and 2 mM glutamine.
    13. Incubate at 37 °C in 5% CO2 for 12-72 h and fix with 4% paraformaldehyde in PBS overnight at 4 °C.

  2. Axon guidance transplant assays
    1. After aspirating MEM in Step A12, use a 45° blade or microscissors to dissect tissue from the donor section (Figure 1F).
      Note: Cutting the tissue explant into an asymmetrical shape will make it easier to maintain orientation during transplant.
    2. Cut a similarly sized piece of tissue from the host section and remove using a pipette.
    3. Using a pipette transfer the donor tissue to the host section and maneuver into position using forceps (Figure 1G).
      Note: To ensure donor axon outgrowth into host tissue, the donor explant must be flush against the host tissue with no visible gap.
    4. Add neurobasal medium and culture for up to 72 h as above to allow for axon outgrowth.
    5. Fix using 4% paraformaldehyde.
    6. Cryoprotect by immersing sections in 30% sucrose in PBS overnight, or until the tissue has sunk, and embedded in 50% OCT embedding matrix; 50% Sucrose/PBS for cryosectioning. Frozen sections can then be processed for immunofluorescence.
    7. If donor tissue expresses GFP or another fluorescent marker, immunofluorescence can then be performed to detect donor axon growth in host tissue.
      Note: While it may be possible to detect fluorescently labeled axons without any further staining, immunofluorescence will give a clearer signal for imaging.
    8. Stained sections can be imaged using epifluorescence or confocal microscopy.

  3. Bead culture assays
    1. Affi-Gel blue gel beads (50 µl) are pre-soaked in recombinant protein or 5 mg/ml bovine serum albumin (BSA) overnight at 4 °C.
      Note: The concentration of recombinant protein used to soak the beads should be determined for each protein used. For our experiments using FGF8 and FGF17, a concentration of 100 µM was used.
    2. After aspirating MEM in Step A12, a pipette can be used to place a single bead onto the tissue section, forceps can then be used to embed the bead into the tissue (Figure 1H).
      Note: Pre-soaked beads can be pipetted into a 60 mm Petri dish filled with PBS to allow easier selection of single beads.
    3. Add neurobasal medium and culture for up to 72 h as above.
    4. Fix using 4% paraformaldehyde in PBS overnight at 4 °C.


      Figure 1. Preparation of tissue sections for culture. A. The dissected brain should be embedded in low melting point agarose, which should be stirred vigorously using a pipette tip. B. Once set, agarose should be trimmed as shown. C. Trimmed agarose block attached to specimen disk using a strong adhesive. D and E. After vibratome sectioning tissue sections should be transferred to floating Whatman Nuclepore membranes using a flat-ended spatula. F and G. For transplant cultures tissue should be cut out from the host section in an asymmetric shape (in this example a ‘wedge’ shape) to make preservation of tissue orientation easier (arrow, F). A similarly shaped piece of tissue from the donor section can then be positioned in the hole left in the host section (arrowhead F and G). H. For bead cultures pre-soaked Affi-Gel blue gel beads can be embedded in tissue as shown.

  4. Immunofluorescence
    1. Cryoprotect tissue using 30% sucrose in PBS overnight, or until the tissue has sunk, and embedded in 50% OCT embedding matrix; 50% Sucrose/PBS for cryosectioning.
    2. Section tissue using a cryostat at a thickness of 10 µm and collect sections on Superfrost Plus slides.
    3. If using an antibody for a morphogen (such as FGF8) perform a methanol fixation step by immersing slides in ice-cold 100% methanol for 30 min.
    4. Permeabilize sections by washing with 0.1% Triton X-100 in 1x PBS for 5 min.
      Note: All wash steps should be performed at room temperature in a Coplin jar or similar and with agitation.
    5. Draw around tissue sections using a hydrophobic barrier pen.
    6. Block sections using a blocking solution of 1% bovine serum albumin in PBS/0.1% Triton X-100, 200 µl per slide, for 1 h at room temperature.
    7. Apply primary antibody diluted in blocking solution, 200 µl per slide, overnight at 4 °C.
      Note: Antibody concentration should be determined by the user, for GFP immunofluorescence used in Figure 2 the anti-GFP antibody (see reagents) was diluted 1 in 300.
    8. Wash with PBS/0.1% Triton X-100 for 5 min.
    9. Apply the appropriate fluorescent conjugated secondary antibody diluted in blocking solution, 200 µl per slide, for 1 h at room temperature.
      Note: Secondary antibody should be raised against the species of the primary antibody used in Step D7. For the GFP immunofluorescence used in Figure 2, a donkey anti-rabbit 488 antibody was used diluted 1 in 200. 
    10. Wash with PBS/0.1% Triton X-100, 5 min.
    11. Counterstain with DAPI diluted 1 in 1,000 in PBS, 10 min at room temperature.
    12. Wash with PBS, 5 min.
    13. Coverslip slides using Vectashield Hardset and leave to dry at room temperature for 1 h.
    14. Once dried, slides can be imaged using epifluorescence or confocal microscopy.


      Figure 2. Axon guidance transplant assay. A and B. An example of a corpus callosum axon guidance assay after immunofluorescence for GFP. An explant of cortical tissue from a GFP expressing donor section has been transplanted into a non-GFP expressing host section. After culture GFP expressing callosal axons have grown from the donor explant and crossed the host midline to reach the opposite cerebral hemisphere (arrows A and B). A and C. Schematic diagram illustrating the transplant experiment. B is a higher magnification image of the boxed region in A. Scale bars = 200 µm.

Recipes

  1. 10x Krebs Buffer
    NaCl
    73.6 g
    126 mM
    KCl
    87 g
    2.5 mM
    NaH2PO4
    1.66 g
    1.2 mM
    CaCl2
    3.68 g
    2.1 mM
    MgCl2
    2.44 g
    1.2 mM
    ddH2O
    1 L

  2. 1x Krebs with antibiotic
    1x Krebs 49 ml
    1 M HEPES 0.5 ml
    Pen-Strep 0.5 ml
    Gentamicin 0.1 ml
  3. MEM with serum
    MEM 44 ml
    FBS 5 ml
    50% Glucose 0.5 ml
    Pen-Strep 0.5 ml
  4. Neurobasal
    Neurobasal 47.5 ml
    B-27 supplement 1 ml
    50% Glucose 0.5 ml
    Pen-Strep 0.5 ml
    Glutamine 0.5 ml

Acknowledgments

This work was supported by funding from the Wellcome Trust, the Biotechnology and Biological
Sciences Research Council and The Simons Initiative for the Developing Brain.

Competing interests

The authors declare no competing financial interests.

Ethics

All mice were bred in-house according to Home Office UK legislation and licenses approved by the University of Edinburgh Ethical Review Committees and Home Office. Animal husbandry was in accordance with UK Animals (Scientific Procedures) Act of 1986 regulations.

References

  1. Clegg, J. M., Parkin, H. M., Mason, J. O. and Pratt, T. (2019). Heparan sulfate sulfation by Hs2st restricts astroglial precursor somal translocation in developing mouse forebrain by a non-cell-autonomous mechanism. J Neurosci 39(8): 1386-1404.
  2. Niquille, M., Garel, S., Mann, F., Hornung, J. P., Otsmane, B., Chevalley, S., Parras, C., Guillemot, F., Gaspar, P., Yanagawa, Y. and Lebrand, C. (2009). Transient neuronal populations are required to guide callosal axons: a role for semaphorin 3C. PLoS Biol 7(10): e1000230.
  3. Pratt, T., Sharp, L., Nichols, J., Price, D. J. and Mason, J. O. (2000). Embryonic Stem Cells and Transgenic Mice Ubiquitously Expressing a Tau-Tagged Green Fluorescent Protein. Dev Biol 28(1): 19-28.

简介

[摘要]器官型切片培养是探索哺乳动物大脑胚胎发育的有力技术。在这个方案中,我们描述了一种基本的切片培养技术,我们已经用于两组实验:轴突引导移植实验和珠子培养实验。

[背景] 器官型切片培养是近年来广泛应用的一种技术,在神经发育领域已成为一种特别流行的技术。它的最大优点是可以在体外培养发育中的神经细胞,同时保持组织的体内结构。在该方案中,我们描述了我们在最近的论文(Clegg等人,2019)中用于两个实验的切片培养技术。首先,我们进行了轴突引导移植实验,将荧光标记的组织移植到非荧光宿主切片上,观察轴突的生长情况。这是先前用于研究胼胝体发育的一个改进版本,但可以很容易地用于检查其他轴突束,如丘脑皮质束(Niquille et al.,2009)。其次,我们进行了微珠培养实验,将浸有重组FGF蛋白的小球植入组织中,检测组织对FGF蛋白的分子和细胞反应。以这种方式使用浸过蛋白的小球提供了一种在特定位置聚焦输送重组蛋白的方法,与将所有组织均匀暴露于蛋白质的浴敷法不同。这模拟了体内的情况,即形态发生素(如FGF17)在特定的解剖位置表达,然后在组织中扩散。该技术可用于探索对多种不同重组蛋白或药物的反应。

关键字:器官型, 切片培养, 移植, 轴突导向, 成形素, 珠子培养

材料和试剂


 


材料


1.     7 ml bijou管(Greiner,目录号:189170)


2.     移液管头(Greiner,目录号:739288)


3.     1.5 ml微型离心管(Greiner,目录号:616201)


4.     Peel-A-Way®嵌入模具(方形–S22,Polysciences)


5.     Falcon®60 mm TC处理中心井器官培养皿(康宁,目录号:353037)


6.     13mm核孔聚碳酸酯径迹蚀刻膜(Whatman,目录号:110401)


7.     试样盘


8.     胶粘剂


 


动物


在我们最初的研究中使用的小鼠普遍表达τGFP,并且已经在前面描述过(Pratt等人,2000)。所有的老鼠都被保存在CD-1背景下(Charles River,菌株代码:022)。用于定时交配的小鼠年龄在6到24周之间。


 


试剂


1.     Earls平衡盐溶液(EBSS)钙、镁、酚红(Thermo Fisher,目录号:24010043)


2.     Seaplalle GTG琼脂糖(Lonza,目录号:50110)


3.     Affi-Gel蓝色凝胶珠(Bio-Rad,目录号:1537301)


4.     MEM(赛默飞世尔,目录号:11090081)


5.     神经基础培养基(Thermo Fisher,目录号:21103049)


6.     1M HEPES(赛默飞世尔,目录号:15630080)


7.     青霉素链霉素(Thermo Fisher,目录号:15140122)


8.     D-葡萄糖溶液(默克公司,目录号:G8769)


9.     庆大霉素50 mg/ml(赛默飞世尔,目录号:15750060)


10.  谷氨酰胺200mm(赛默飞世尔,产品目录号:25030081)


11.  胎牛血清(默克公司,目录号:F4135)


12.  B-27增补件(赛默飞世尔,目录号:17504044)


13.  OCT嵌入矩阵(Cellpath,目录号:KMA-0100-00A)


14.  1个Krebs缓冲液(10倍库存,见配方)


15.  1x PBS(赛默飞世尔产品目录号:14190094)


16.  NaCl(赛默飞世尔产品目录号:10428420)


17.  KCl(赛默飞世尔产品目录号:10684732)


18.  NaH2PO4(赛默飞世尔产品目录号:10723621)


19.  CaCl2(赛默飞世尔产品目录号:10657662)


20.  MgCl2(赛默飞世尔产品目录号:10386743)


21.  Triton X-100(默克公司,目录号:10789704001)


22.  牛血清白蛋白(默克公司,目录号:A1933)


23.  抗GFP抗体(Abcam,目录号:ab290)


24.  山羊抗兔IgG 488(赛默飞世尔,目录号:A-11008)


25.  DAPI(赛默飞世尔,目录号:D1306)


26.  Vectashield Hardset(VectorLabs,目录号:H-1400)


27.  10倍Krebs缓冲液(见配方)


28.  1x含抗生素的Krebs(见配方)


29.  含血清的MEM(见配方)


30.  神经基础(见食谱)


设备


 


1.     微量加样器


2.     层流组织培养罩


3.     组织培养箱(37°C,5%CO2)


4.     解剖显微镜(目录号:MEDZ1100)


5.     徕卡VT1000 S振动仪


6.     水浴


7.     带平端叶片的Fisherbrand微型平台(Thermo Fisher,目录号:21-401-20)


8.     镊子,尺寸5(精细科学工具,目录号:11251-20)


9.     5 mm切削刃微型切割机(精细科学工具,目录号:15003-08)


10.  剪刀,9厘米(精细科学工具,目录号:14060-09)


11.  45°无菌刀片(Altomed,型号:A10136)


12.  低温恒温器(徕卡,目录号:CM3050 S)


13.  Super frost Plus幻灯片(赛默飞世尔,目录号:J1800AMNZ)


14.  疏水阻隔笔(VectorLabs,目录号:H-4000)


 


程序


 


A、 器官型切片培养


1.     通过使100%的氧气鼓泡通过溶液直到观察到颜色变化(粉红色到橙色)来充氧EBSS。然后将电子束放在冰上冷却。


2.     通过颈部脱臼或过量使用麻醉剂,在理想的胚胎期淘汰怀孕的母鼠。


注:切片培养可从胚胎期(E)13.5日起进行。对于轴突引导分析,应选择一个阶段,在这一阶段,轴突从被调查的神经束中生长出来,例如胼胝体的E17.5。


3.     取下子宫,放在一个冰上的预冷充氧EBSS盘中,在组织培养罩中,用剪刀和镊子从子宫中取出胚胎。用剪刀把胚胎的头部从身体上剪下来,然后转移到第二盘EBSS上。


4.     用镊子从头部剥下皮肤露出头骨,用显微顺时针切下颅骨中线,剥回颅骨露出大脑。用镊子小心地把大脑从脑底分离出来。


注意:在怀孕的母鼠被宰杀后的1小时内,大脑应该被解剖出来并放置在EBSS中。


5.     收集7毫升装有充氧EBS的bijou试管中的大脑,并将试管放在冰上。


注意:在这个阶段,大脑可以保持3小时,以便有时间进行基因分型(如有必要)。如果计划在这个阶段保存大脑,建议在EBSS中添加葡萄糖(浓度为0.1%)。


6.     将大脑分别包埋在大约5毫升的低熔点琼脂糖中。使用前,琼脂糖应在42°C的温度下使用水浴。一旦组织就位,将模具放在冰上(图1A)。


注意:在将大脑定位到所需平面之前,反复搅拌组织周围的琼脂糖,以清除组织中残留的EBS。这样可以防止组织在切片过程中与琼脂糖分离(图1A)。


7.     一旦凝固,从模具中取出琼脂糖,并用剃刀刀片修剪,直到含有组织的琼脂糖块<1 cm3(图1B)。使用粘合剂将琼脂糖块连接到样本盘上(图1C)。


注意。切片平面应根据受检脑区或轴突束确定,胼胝体轴突导向分析采用冠状切面,并收集含有端脑的组织切片。


8.     在冷1x Krebs缓冲液中以400µm的截面厚度切割振动棒上的组织。


注意:剖切时,可控震源应设置为频率设置为8,速度设置为4。如果组织开始破裂,减速至3。


9.     收集含有100 mM HEPES、0.5 mg/ml庆大霉素和1%青霉素链霉素的1x Krebs组织切片。


注意:组织切片的转移应始终使用钝头小孢子,不应使用刷子,因为组织可能粘在刷毛上造成损伤。


10.  在器官培养皿中加入1ml含血清的MEM。将一个Whatman核孔膜放在MEM上,在孔的中心(有光泽的一面面向液体)。


11.  小心地将单个组织切片转移到膜的中心,确保膜的任何部分都不会被淹没(图1D和1E)。在37°C和5%CO2中培养1小时。


12.  抽吸MEM,用含有2%B27补充剂、1%青霉素链霉素、0.5%葡萄糖和2mm谷氨酰胺的1ml神经基础培养基代替。


13.  在温度为4℃、温度为4℃、温度为4℃、温度为37℃、温度为4.5%的甲醛溶液中培养过夜。


 


B、 轴突引导移植试验


1.     在步骤A12中吸出MEM后,使用45°刀片或微顺槽从供体部分剥离组织(图1F)。


注意:将组织外植体切割成不对称的形状会使移植过程中更容易保持定向。


2.     从宿主部分切一块大小相似的组织,用移液管移走。


3.     使用移液管将供者组织转移到宿主部分,并使用镊子移动到位(图1G)。


注:为确保供者轴突生长到宿主组织中,供体外植体必须与宿主组织齐平,没有可见的间隙。


4.     加入神经基础培养基,培养72小时,使轴突生长。


5.     用4%多聚甲醛固定。


6.     冷冻保护:将切片浸入30%蔗糖PBS中过夜,或直到组织下沉,并嵌入50%OCT包埋基质中;50%蔗糖/PBS用于冷冻切片。冷冻切片可以进行免疫荧光处理。


7.     如果供体组织表达GFP或其他荧光标记物,免疫荧光可检测宿主组织中供体轴突的生长情况。


无荧光标记的轴突可作进一步的荧光标记。


8.     染色切片可以用荧光显微镜或共焦显微镜成像。


 


C、 珠子培养试验


1.     将Affi-Gel蓝色凝胶珠(50µl)预浸在重组蛋白或5 mg/ml牛血清白蛋白(BSA)中,在4℃下过夜。


注:用于浸泡珠子的重组蛋白的浓度应根据所使用的每种蛋白质来确定。在我们使用FGF8和FGF17的实验中,使用了100µM的浓度。


2.     在步骤A12中吸出MEM后,可使用吸管将单个珠子放在组织切片上,然后使用镊子将珠子嵌入组织中(图1H)。


注:预先浸过的珠子可以用移液管移入装满PBS的60 mm培养皿中,以便更容易地选择单个珠子。


3.     加入神经基础培养基,培养72小时,如上所述。


4.     使用4%多聚甲醛在PBS中于4°C下过夜固定。


 






图1。培养用组织切片的制备。A、 解剖后的大脑应植入低熔点琼脂糖中,用吸管尖用力搅拌。B、 一旦凝固,琼脂糖应该修剪如图所示。C、 用强力粘合剂将切好的琼脂糖块附在标本盘上。D和E。振动切片后,应使用平头刮刀将组织切片转移到漂浮的Whatman核孔膜上。F和G。对于移植培养物,应以不对称的形状(在本例中为“楔形”形状)从宿主部分切下组织,以便于保存组织方向(箭头,F)。然后,可以将来自供体部分的类似形状的组织块放置在宿主部分的左侧孔中(箭头F和G)。H、 对于珠子培养,预先浸泡的Affi凝胶蓝色凝胶珠可以嵌入组织中,如图所示。


 


D、 免疫荧光


1.     在PBS中使用30%蔗糖冷冻保护组织过夜,或直到组织下沉,并嵌入50%OCT包埋基质中;50%蔗糖/PBS用于冷冻切片。


2.     使用厚度为10µm的低温恒温器切片,并在Superfrost Plus载玻片上收集切片。


3.     如果使用吗啉原抗体(如FGF8),则通过将载玻片浸入100%冰甲醇中30分钟来进行甲醇固定步骤。


4.     通过在1x PBS中用0.1%Triton X-100清洗5分钟,使部分渗透。


注意:所有清洗步骤应在室温下在Coplin罐或类似容器中进行,并搅拌。


5.     用疏水性阻隔笔画出组织切片。


6.     在室温下,使用1%牛血清白蛋白PBS/0.1%Triton X-100,200µl/玻片的封闭溶液封闭切片1小时。


7.     在封闭溶液中稀释一级抗体,每片200µl,在4°C下过夜。


注:抗体浓度由用户确定,图2中使用的GFP免疫荧光法,抗GFP抗体(见试剂)稀释1:300。


8.     用PBS/0.1%Triton X-100清洗5分钟。


9.     将适当的荧光结合二级抗体在封闭溶液中稀释,每片200µl,在室温下放置1小时。


注:应针对步骤D7中使用的一级抗体的种类提高二级抗体。对于图2中使用的GFP免疫荧光,使用驴抗兔488抗体稀释1:200。


10.  用PBS/0.1%Triton X-100清洗5分钟。


11.  用稀释1:1000的磷酸二铵在PBS中复染,室温下10分钟。


12.  用PBS清洗,5分钟。


13.  使用Vectashield硬套覆盖滑片,并在室温下晾干1小时。


14.  一旦干燥,载玻片可以用荧光显微镜或共焦显微镜成像。


 






图2。轴突引导移植试验。A和B。GFP免疫荧光后胼胝体轴突导向试验的一个例子。从表达绿色荧光蛋白的供体部分的皮质组织的外植体被移植到不表达绿色荧光蛋白的宿主部分。培养后,表达GFP的胼胝体轴突从供体外植体生长,穿过宿主中线到达对侧大脑半球(箭头A和B)。A和C。移植实验示意图。B是a中框状区域的高倍图像。比例尺=200µm。


食谱


 


1.     10倍Krebs缓冲器


氯化钠73.6克126毫米


KCl 87克2.5毫米


NaH2PO4 1.66克1.2毫米


氯化钙3.68克2.1毫米


氯化镁2.44克1.2毫米


ddH2O 1升


2.     1x含抗生素的Krebs


1x Krebs 49毫升


1 M HEPES 0.5毫升


链球菌0.5毫升


庆大霉素0.1毫升


3.     含血清的MEM


内存44毫升


FBS 5毫升


50%葡萄糖0.5ml


链球菌0.5毫升


4.     神经基底


神经基底47.5毫升


B-27补充1毫升


50%葡萄糖0.5ml


链球菌0.5毫升


谷氨酰胺0.5毫升


 


致谢


 


这项工作得到了Wellcome信托基金、生物技术和生物技术的资助


科学研究委员会和西蒙斯大脑发展倡议。


 


相互竞争的利益


 


作者声明没有竞争性的经济利益。




 


伦理学


 


所有的老鼠都是根据英国内政部的立法和爱丁堡大学伦理审查委员会和内政部批准的执照在室内饲养的。畜牧业是根据英国动物(科学程序)法1986年的规定。


 


工具书类


 


1.     Clegg,J.M.,Parkin,H.M.,Mason,J.O.和Pratt,T.(2019年)。硫酸乙酰肝素硫酸化对小鼠前脑星形胶质前体转位的非细胞自主机制的影响。神经病学杂志39(8):1386-1404。


2.     Niquille,M.,Garel,S.,Mann,F.,Hornung,J.P.,Otsmane,B.,Chevalley,S.,Parras,C.,Guillemot,F.,Gaspar,P.,Yanagawa,Y.和Lebrand,C.(2009年)。短暂的神经元群需要引导胼胝体轴突:信号蛋白3C的作用。公共科学图书馆生物学7(10):e1000230。


3.     Pratt,T.,Sharp,L.,Nichols,J.,Price,D.J.和Mason,J.O.(2000年)。胚胎干细胞和转基因小鼠普遍表达Tau标记的绿色荧光蛋白。28(1):19-28。求文献一篇
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Clegg, J. M. and Pratt, T. (2020). Organotypic Slice Culture of the Embryonic Mouse Brain. Bio-protocol 10(13): e3674. DOI: 10.21769/BioProtoc.3674.
  2. Clegg, J. M., Parkin, H. M., Mason, J. O. and Pratt, T. (2019). Heparan sulfate sulfation by Hs2st restricts astroglial precursor somal translocation in developing mouse forebrain by a non-cell-autonomous mechanism. J Neurosci 39(8): 1386-1404.
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