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Olfactory Bulb (OB) Transplants
大脑嗅球(OB)移植   

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Abstract

Transplantation in mouse brain slices is a powerful tool in order to study axon targeting and migrational events during development. Taking advantage of donors and recipients belonging to different genotypes, this technique allows researchers to assess the contribution of donor and/or recipient tissue by performing various combinations and to study cell-autonomous functions or effects that are influenced by the recipient’s environment (Bastakis, et al., 2015). Here we describe the transplantation procedure on sagittal brain slices containing olfactory bulb (OB). Specifically, we have transplanted the proximal-to-the-cortex part of dorsal OB to the same region on a recipient slice. Transplanted slices can be cultured for up to 3 days before their morphology is disfigured due to growth in 3D. Re-sectioning of these slices allows for a more detailed immunohistochemical analysis.

Materials and Reagents

  1. Millicell cell culture insert, 30 mm, hydrophilic PTFE, 0.4 µm (Merck Millipore, catalog number: PICM0RG50 )
  2. Disposable transfer pipettes (SARSTEDT, catalog number: 86.1172.001 )
  3. 5 ml stripette disposable serological pipette (Sigma-Aldrich, catalog number: CLS4051 )
  4. 50 ml Falcon tube
  5. Sterile 24-well plate
  6. Stainless surgical blade (Swann Morton, Lance, model: No. 24 )
  7. Vibratome blade (Campden Instruments, catalog number: 752-1-SS )
  8. 60 mm culture dishes
  9. Straight triangle insert pin (Fine Science Tools, catalog number: 26007-04 )
  10. Stainless steel minutien pins 0.10 mm (Fine Science Tools, catalog number: 26002-10 )
  11. Filters (0.2 μm, 0.45 μm)
  12. Mouse embryos at embryonic days E13.5-E15.5
  13. Leibovitz’s L-15 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11415-064 )
  14. SeaPlaqueTM low melting temperature agarose (Lonza, catalog number: 50101 )
  15. Sterile water
  16. 100 units/ml penicillin-streptomycin (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15070-063 )
  17. NaCl (Merck Millipore, catalog number: 106404 )
  18. KCl (Merck Millipore, catalog number: 104936 )
  19. Na2HPO4 (Merck Millipore, catalog number: 106586 )
  20. NaH2PO4 (Sigma-Aldrich, catalog number: S-0751 )
  21. NaOH (Merck Millipore, catalog number: 106498 )
  22. Glucose (Sigma-Aldrich, catalog number: G8270 )
  23. DMEM/F12 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21041-025 )
  24. Fetal bovine serum (FBS) (Merck Millipore, Biochrom, catalog number: S0115 )
  25. 1x glutamax (Thermo Fisher Scientific, GibcoTM, catalog number: 35050-038 )
  26. N-2 supplement (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 17502-048 )
  27. Neurobasal medium (Thermo Fisher Scientific, GibcoTM, catalog number: 12348-017 )
  28. B-27 supplement (50x) (Thermo Fisher Scientific, GibcoTM, catalog number: 17504-044 )
  29. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
  30. Leibovitz’s L-15/penicillin-streptomycin medium (see Recipes)
  31. 3% SeaPlaque agarose in Leibovitz’s L-15 (see Recipes)
  32. 10x phosphate buffer solution (PBS) (see Recipes)
  33. 10 M NaOH (see Recipes)
  34. Sterile 1x PBS (0.1 M PBS) with 100 units/ml penicillin-streptomycin (see Recipes)
  35. 25% glucose in 1x PBS (see Recipes)
  36. DMEM/F12 plus medium (see Recipes)
  37. Neurobasal/ B-27 plus medium (See Recipes)
  38. 4% paraformaldehyde (PFA) (see Recipes)

Equipment

  1. 37 °C, 5% CO2 forced-air incubator (Thermo Fisher Scientific, model: 3110 )
  2. Dissecting microscope (Leica Microsystems, model: MZ125 )
  3. 2 Dumont #5 forceps (Fine Science Tools, catalog number: 11251-10 )
  4. Moria perforated spoon (Fine Science Tools, catalog number: 10370-17 )
  5. 250 ml glass beaker
  6. Two-in-One micro spatula (Fine Science Tools, catalog number: 10091-12 )
  7. Leica vibratome (VT 1000S)
  8. Nickel plated pin holder (Fine Science Tools, catalog number: 26018-17 )

Note: All surgical tools should be UV-sterilized before use.

Procedure

Housing and all animal procedures used were according to the European Union policy (Directive 86/609/EEC) and institutionally approved protocols. E0.5: day of the vaginal plug.

  1. Pregnant mice of recipient and donor genotypes are sacrificed with cervical dislocation at the same day of gestation. Embryos at embryonic day 14.5 (E14.5) are easier to handle.
  2. Embryos are removed from their yolk sacs and their brain is dissected under a dissecting microscope in Leibovitz’s L-15/penicillin-streptomycin medium using Dumont #5 forceps (Video 1).

    Video 1. Brain dissection from a mouse E14.5 embryo. The video illustrates step by step instructions for the dissection of the brain from a mouse embryo at the embryonic stage E14.5.

  3. Cortices and OBs are separated from the midbrain (Video 1). An illustration of the cortices and OBs as well as the site of the transplant is shown in Figure 1. We add 2 ml of pre-warmed 3% SeaPlaque agarose in Leibovitz’s/L-15 (60 °C) per well of a 24-well plate placed on ice. Each sample is transferred with a perforated spoon as shown in Figure 2A.


    Figure 1. Illustration showing a top and side view of the forebrain from a mouse E14.5 embryo. The area of transplantation is indicated in red.

  4. Swirl the liquid using a 200 μl pipette tip to prevent the sample from touching the bottom of the well (Figure 2B). It is preferable that you set the orientation of your sample in the agarose with the tip before it solidifies, after approximately 10-15 min. In our case the olfactory bulbs are facing up in a vertical position.


    Figure 2. Embedding of a tissue in 3% SeaPlaque agarose in Leibovitz’s L-15 on ice. A. A dissected brain is transferred with a perforated spoon in a well with 3% SeaPlaque agarose in Leibovitz’s L-15. The 24-well plate with the agarose has previously been put on ice. B. Swirl the liquid using a 200 μl pipette tip. Without disrupting the tissue, orient it with the tip on a vertical position. Swirl for approximately 10 min until the agarose starts solidifying.

  5. Fill the tank of the vibratome with ice-cold 1x PBS with penicillin-streptomycin.
  6. Use a new UV-sterilized vibratome blade.
  7. Remove the agarose block containing the tissue from the plastic well using a micro spatula.
  8. Cut the agarose block with a steel blade in order to create a trapezoid with the tissue placed horizontally in its center (Figure 3A).
  9. Use instant glue and fix the tissue block to the metallic specimen plate of the vibratome with the big base of the trapezoid facing down.
  10. Adapt the specimen plate in the vibratome and set the speed to 5, the frequency to 5 and the feed to 250 μm.
  11. Perform continuous cutting and transfer the sections that contain the olfactory bulb to a 60 mm culture dish containing 2 ml of Leibovitz’s L-15/penicillin-streptomycin medium, using a perforated spoon (Figure 3B).
  12. Put the cell culture insert with the correct orientation in a new 60 mm culture dish containing 2.2 ml of DMEM/F12 plus medium. On the top surface of the membrane add 100 μl of DMEM/F12 plus medium in spots as shown in Figure 3C.
  13. Remove the surrounding agarose from the tissue sections using the dissecting forceps and transfer them on top of the cell culture insert membrane with a sterile disposable transfer pipette (Figure 3D). Use separate membranes for the recipient and the donor sections.
  14. Incubate at 37 °C, 5% CO2 for 1 h.
  15. Take the dish out of the incubator and remove 2 ml of the medium from the plate, using a 5 ml sterile disposable pipette. Afterwards, add 2 ml of prewarmed (at 37 °C) Neurobasal/B27 plus medium to the plate. The sections on top of the membrane remain wet during the whole procedure, and their medium will be gradually replaced with the new one through the membrane pores that are in contact with it.
  16. Under the dissecting microscope cut a small triangular piece of the OB in the recipient section using the straight triangle insert pin (adapted to the Pin Holder) and remove it from the section with a 200 μl pipette.
  17. Cut a slightly bigger piece of the donor OB section and transfer it with a 200 μl pipette (transplant in medium) close to the recipient section (Figure 3E).
  18. Push the transplant (Red* in Figure 3E) towards the missing part of the recipient and adjust it using a minutien pin (adapted to the pin holder) (Figure 3F).
  19. Incubate the transplanted sections (Figure 3F) at 37 °C, 5% CO2 for 24, 48 or 72 h. Change medium every 24 h.


    Figure 3. Transplantation procedure in steps. A. A trapezoid is prepared with the tissue placed horizontally in its center. B. Sagittal section containing the forebrain. C. A cell culture insert is applied in a 60 mm culture dish containing 2.2 ml of DMEM/F12 plus medium. Add in spots 100 μl of DMEM/F12 plus medium. D. A brain section, clean of agarose, on the top surface of the cell culture insert. E. The proximal-to-the-cortex part of dorsal OB has been removed from the recipient slice and a slightly larger piece from the same site of the donor is transferred close to the recipient (red asterisk). F. The transplant has been applied to the recipient slice with the correct orientation. Scale: 1 mm in D, E, F.

  20. For further sectioning fix the tissue section with 4% PFA for 1 h at RT, wash in 1x PBS and transfer it to the bottom of a clean well in a 24-well plate.
  21. Dry the section and re-embed it in 3% of SeaPlaque agarose as described before (the section now should stay at the bottom of the well).
  22. Re-section the tissue in a similar way to the one described before in the vibratome at a 50 μm thickness.
  23. Continue with immunostaining as described in Bastakis et al. (2015).

Representative data

Representative images of the expected results regarding migration of projection neurons (Tbr1+) from the proximal-to-the-cortex part of dorsolateral OB derived from a GFP-labelled mouse (BrdU pulsed at E11.5) is shown below (Figure 4, adapted from Bastakis et al., 2015).


Figure 4. Transplantation of dorsal OB from mouse brain sections for the study of migrating projection neurons. A. Transplants were generated on sagittal E14.5 sections as shown in the scheme, with donor cells expressing GFP or being BrdU-pulsed at E11.5. B. WT GFP+, BrdU+ (BrdU injection at E11.5) cells were transplanted on WT recipients and immunostained for GFP and Tbr1 (marker of immature OB projection neurons) or GFP and BrdU. Migrating cells (GFP+) were projection neurons (co-localization with Tbr1, arrowheads, upper images) and were born at E11.5 (co-localization with BrdU, arrowheads, lower images). The dorsal part of the slice is shown in B. Scale: 150 μm.

Recipes

  1. Leibovitz’s L-15/penicillin-streptomycin (50 ml)
    0.5 ml 100x penicillin-streptomycin
    49.5 ml Leibovitz’s L-15
    Filter sterilize (0.2 μm)
    Store at 4 °C
  2. 3% SeaPlaque agarose in Leibovitz’s L-15 (10 ml)
    1. Use the microwave at high power to warm up 10 ml of Leibovitz’s L-15 medium in a 50 ml Falcon tube until bubbles appear (just before boiling).
      Note: The Falcon tube should be sunk in a 250 ml beaker containing 100 ml of water and the cup should be slightly loose.
    2. Dissolve 0.3 g of SeaPlaque agarose in the pre-warmed medium by vigorous shaking. If there are still undissolved particles, boil for 1 more min as previously described.
    3. Cool the solution in a water bath to 60 °C prior to use.
  3. 10x PBS (1 liter)
    Mix 70.1 g of NaCl with 2 g of KCl, 12.8 g of Na2HPO4, 4.4 g of NaH2PO4
    If necessary, adjust pH to 7.4 with 10 M NaOH
    Add dH2O to 1 liter
    Filter sterilize (0.2 μm)
    Store at RT
  4. 10 M NaOH
    Weight 40 g of NaOH in a beaker
    Add dH2O to make solution up to 100 ml
    Store at RT
  5. 1x PBS with 100 units/ml penicillin-streptomycin
    Add 100 ml of 10x PBS to 890 ml of sterile water and mix
    Add 10 ml of 100x penicillin-streptomycin stock
    Mix and filter sterilize (0.2 μm)
    Store at 4 °C
  6. 25% glucose in 1x PBS (50 ml)
    Dissolve 12.5 g of glucose in 30 ml of 1x PBS
    Add 1x PBS to 50 ml
    Filter sterilize (0.2 μm)
    Store at 4 °C
  7. DMEM/F12 plus (25 ml)
    0.6 ml 25% glucose
    1.25 ml FBS
    0.25 ml 100x penicillin-streptomycin
    0.25 ml 100x glutamax
    0.25 ml N2 supplement
    22.4 ml DMEM/F12
    Filter sterilize (0.2 μm)
    Store at 4 °C
  8. Neurobasal/B27 plus (50 ml)
    1.2 ml 25% glucose
    0.5 ml 100x penicillin-streptomycin
    0.5 ml 100x glutamax
    1 ml B27 supplement
    47.75 ml neurobasal
    Filter sterilize (0.2 μm)
    Store at 4 °C
  9. 4% paraformaldehyde (PFA) in 1x PBS
    Dissolve 4 g PFA in a bottle with 100 ml of prewarmed 1x PBS (at 65 °C)
    Add 20 μl of 10 M NaOH and mix well until the solution is clear
    Filter with a 0.45 μm filter and store at 4 °C for 1 week or at -20 °C for several months

Acknowledgments

This protocol was adapted from the previously published study, de Diego et al. (2002) and it was performed by Bastakis et al. (2015). This work was supported by the European Commission FP7 programme “Translational Potential” [contract number 285948], InnovCrete [316223], ARISTEIA I [Project 593 MyelinTag] and by Institute of Molecular Biology and Biotechnology intramural grants.

References

  1. Bastakis, G. G., Savvaki, M., Stamatakis, A., Vidaki, M. and Karagogeos, D. (2015). Tag1 deficiency results in olfactory dysfunction through impaired migration of mitral cells. Development 142(24): 4318-4328.
  2. de Diego, I., Kyriakopoulou, K., Karagogeos, D. and Wassef, M. (2002). Multiple influences on the migration of precerebellar neurons in the caudal medulla. Development 129(2): 297-306.

简介

小鼠脑切片中的移植是一个强大的工具,以便研究轴突靶向和发展过程中的迁移事件。 利用属于不同基因型的供体和受体,该技术允许研究人员通过进行各种组合来评估供体和/或受体组织的贡献,并研究受受体环境影响的细胞自主功能或效应(Bastakis, em>等人,2015)。 在这里我们描述的矢状大脑切片包含嗅球(OB)的移植程序。 具体来说,我们已经将背侧OB的近端到外皮部分移植到受体切片上的相同区域。 移植的切片可以培养长达3天,之后由于3D中的生长它们的形态被破坏。 这些切片的重新切片允许更详细的免疫组织化学分析。

材料和试剂

  1. Millicell细胞培养插入物,30mm,亲水性PTFE,0.4μm(Merck Millipore,目录号:PICM0RG50)
  2. 一次性移液器(SARSTEDT,目录号:86.1172.001)
  3. 5ml Sigma-Aldrich一次性血清移液管(Sigma-Aldrich,目录号:CLS4051)
  4. 50ml Falcon管
  5. 无菌24孔板
  6. 材料和试剂

    1. Millicell细胞培养插入物,30mm,亲水性PTFE,0.4μm(Merck Millipore,目录号:PICM0RG50)
    2. 一次性移液器(SARSTEDT,目录号:86.1172.001)
    3. 5ml Sigma-Aldrich一次性血清移液管(Sigma-Aldrich,目录号:CLS4051)
    4. 50ml Falcon管
    5. 无菌24孔板
    6. ...

      材料和试剂

      1. Millicell细胞培养插入物,30mm,亲水性PTFE,0.4μm(Merck Millipore,目录号:PICM0RG50)
      2. 一次性移液器(SARSTEDT,目录号:86.1172.001)
      3. 5ml Sigma-Aldrich一次性血清移液管(Sigma-Aldrich,目录号:CLS4051)
      4. 50ml Falcon管
      5. 无菌24孔板
      6. ......

      7. (Merck Millipore,目录号:106586)
      8. NaH 2 PO 4(Sigma-Aldrich,目录号:S-0751)
      9. NaOH(Merck Millipore,目录号:106498)
      10. 葡萄糖(Sigma-Aldrich,目录号:G8270)
      11. DMEM/F12培养基(Thermo Fisher Scientific,Gibco TM ,目录号:21041-025)
      12. 胎牛血清(FBS)(Merck Millipore,Biochrom,目录号:S0115)
      13. 1×glutamax(Thermo Fisher Scientific,Gibco TM ,目录号:35050-038)
      14. N-2补充物(100x)(Thermo Fisher Scientific,Gibco TM ,目录号:17502-048)
      15. Neurobasal培养基(Thermo Fisher Scientific,Gibco TM ,目录号:12348-017)
      16. B-27补充(50x)(Thermo Fisher Scientific,Gibco TM ,目录号:17504-044)
      17. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)
      18. Leibovitz的L-15 /青霉素 - 链霉素培养基(参见Recipes)
      19. 在Leibovitz's L-15中的3%SeaPlaque琼脂糖(参见Recipes)
      20. 10x磷酸盐缓冲溶液(PBS)(见配方)
      21. 10 M NaOH(见配方)
      22. 具有100单位/ml青霉素 - 链霉素的无菌1x PBS(0.1M PBS)(参见Recipes)
      23. 25%葡萄糖的1x PBS(见配方)
      24. DMEM/F12加培养基(见配方)
      25. Neurobasal/B-27加培养基(见配方)
      26. 4%多聚甲醛(PFA)(参见配方)

      设备

      1. 37℃,5%CO 2强制空气培养箱(Thermo Fisher Scientific,型号:3110)中。
      2. 解剖显微镜(Leica Microsystems,型号:MZ125)
      3. 2 Dumont#5镊子(Fine Science Tools,目录号:11251-10)
      4. Moria穿孔勺(Fine Science Tools,目录号:10370-17)
      5. 250ml玻璃烧杯
      6. 二合一微铲(Fine Science Tools,目录号:10091-12)
      7. 徕卡vibratome(VT 1000S)
      8. 镀镍针座(Fine Science Tools,目录号:26018-17)

      注意:所有手术工具在使用前都应进行紫外线灭菌。

      程序

      所使用的住房和所有动物程序均根据欧盟政策(指令86/609/EEC)和经制度批准的方案。 E0.5:阴道栓塞的一天。

      1. 在妊娠的同一天用子宫颈脱位处死受体和供体基因型的怀孕小鼠。胚胎在胚胎14.5(E14.5)胚胎更容易处理
      2. 从其卵黄囊中取出胚胎,并使用Dumont#5镊子在Leibovitz's L-15 /青霉素 - 链霉素培养基中在解剖显微镜下解剖它们的脑(视频1)。

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      3. 皮层和OB与中脑分离(视频1)。图1显示了皮层和OB以及移植部位的图示。我们在Leibovitz's/L-15(60℃)中的24孔板中加入2ml预热的3%SeaPlaque琼脂糖,孔板置于冰上。每个样品用如图2A所示的多孔勺子转移

        图1.图示显示来自小鼠E14.5胚胎的前脑的顶视图和侧视图。移植区域以红色表示。

      4. 使用200μl移液管尖旋转液体,以防止样品接触孔底部(图2B)。优选地,在大约10-15分钟之后,在凝固之前,使用尖端在琼脂糖中设置样品的取向。在我们的情况下,嗅球是垂直向上的

        图2.将组织包埋在冰上的Leibovitz's L-15中的3%SeaPlaque琼脂糖中。A.用穿孔勺子将解剖的脑转移到含有3%SeaPlaque琼脂糖的孔中的Leibovitz's L- 15。将具有琼脂糖的24孔板预先置于冰上。 B.使用200μl移液管尖旋转液体。在不破坏组织的情况下,使其在垂直位置上定向。旋转约10分钟,直到琼脂糖开始凝固
      5. 填充vibratome的坦克与冰冷的1×PBS与青霉素 - 链霉素。
      6. 使用新的紫外线消毒的vibratome刀片。
      7. 用微量刮刀从塑料孔中取出含有组织的琼脂糖块
      8. 用钢刀片切割琼脂糖块以形成梯形,其中组织水平放置在其中心(图3A)。
      9. 使用瞬间胶水,并将组织块固定到vibratome的金属标本板,梯形的大底朝下。
      10. 适应振动台上的标本板,设置速度为5,频率为5,进给为250μm。
      11. 使用穿孔勺子进行连续切割,并将含有嗅球的切片转移到含有2 ml Leibovitz's L-15 /青霉素 - 链霉素培养基的60 mm培养皿中(图3B)。
      12. 将具有正确方向的细胞培养插入物置于含有2.2ml DMEM/F12加培养基的新的60mm培养皿中。在膜的顶表面上加入100μl的DMEM/F12加培养基,如图3C所示
      13. 使用解剖钳从组织切片中移除周围的琼脂糖,并用无菌一次性移液管将其转移到细胞培养插入膜的顶部(图3D)。对受体和供体部分使用单独的膜
      14. 在37℃,5%CO 2孵育1小时
      15. 取出的培养皿,从培养箱中取出2毫升培养基,使用5毫升无菌一次性吸管从板。然后,向板中加入2ml预热(在37℃)Neurobasal/B27加培养基。膜的顶部的部分在整个程序期间保持湿润,并且它们的介质将通过与其接触的膜孔逐渐替换为新的。
      16. 在解剖显微镜下,使用直的三角形插针(适合于针座)在受体部分切开一个小三角形的OB,并用200μl移液管将其从切片上取下。
      17. 切割一个稍大一点的供体OB部分,并用接近受体部分的200μl移液管(在培养基中移植)转移它(图3E)。
      18. 将移植物(图3E中的红色*)推向接受者的缺失部分,并使用微型针(适合针型支架)进行调整(图3F)。
      19. 在37℃,5%CO 2孵育移植切片(图3F)24,48或72小时。每24小时更换一次培养基。


        图3.步骤中的移植程序 A.准备梯形,其中组织水平放置在其中心。 B.包含前脑的矢状断面。 C.将细胞培养插入物施加在含有2.2ml DMEM/F12加培养基的60mm培养皿中。加入斑点100微升的DMEM/F12加培养基。 D.在细胞培养插入物的顶表面上的脑切片,干净的琼脂糖。 E.背侧OB的近端至皮层部分已经从接受者切片移除,并且来自供体的相同部位的稍大片被转移到接受者附近(红色星号)。 F.移植已经以正确的方向应用于受体切片。刻度:在D,E,F中为1mm
      20. 为了进一步切片,用4%PFA在室温下固定组织切片1小时,在1x PBS中洗涤,并将其转移至24孔板中的干净孔的底部。
      21. 干燥切片并如前所述将其重新嵌入3%的SeaPlaque琼脂糖中(切片现在应该留在孔底部)。
      22. 以类似于前面描述的方式在振动器中以50μm厚度重新切割组织
      23. 继续如Bastakis等人所述的免疫染色。 (2015)。

      代表数据

      关于从来自GFP标记的小鼠(BrdU在E11.5脉冲)的背外侧OB的近端至外皮部分的投影神经元(Tbr1 + )的迁移的预期结果的代表性图像,如下所示(图4,改编自Bastakis等人,2015)。


      图4.来自小鼠脑切片的背部OB的移植用于迁移投影神经元的研究 A.如方案中所示,在矢状E14.5切片上产生移植物,其中供体细胞表达GFP或BrdU脉冲在E11.5。将WT GFP ,BrdU + (在E11.5的BrdU注射)细胞移植到WT受体上,并对GFP和Tbr1(未成熟OB投影神经元的标记)进行免疫染色,或GFP和BrdU。迁移细胞(GFP + )是投影神经元(与Tbr1,箭头,上图像共定位),出生于E11.5(与BrdU,箭头,下图像共定位)。切片的背部显示为B.比例:150μm。

      食谱

      1. Leibovitz's L-15 /青霉素 - 链霉素(50ml) 0.5ml 100x青霉素 - 链霉素 49.5ml Leibovitz's L-15
        过滤灭菌(0.2μm)
        存储在4°C
      2. 3%SeaPlaque琼脂糖在Leibovitz's L-15(10ml)中
        1. 使用高功率微波炉在50毫升Falcon管中加热10毫升Leibovitz's L-15培养基,直到气泡出现(即将煮沸前)。
          注意:Falcon管应该放在装有100毫升水的250毫升烧杯中,杯应稍微松一些。
        2. 通过剧烈振荡将0.3g的SeaPlaque琼脂糖溶解在预热的培养基中。 如果仍有未溶解的颗粒,如前所述再沸腾1分钟。
        3. 使用前将溶液在水浴中冷却至60°C
      3. 10x PBS(1升)
        将70.1g的NaCl与2g的KCl,12.8g的Na 2 HPO 4,4.4g的NaH 2 PO 4
        如果需要,用10M NaOH将pH调节至7.4 将dH <2> O添加到1升
        过滤灭菌(0.2μm)
        在RT存储
      4. 10 M NaOH
        重量40克NaOH在烧杯中
        加入dH 2 O使溶液达到100ml/dm 2 在RT存储
      5. 1×PBS用100单位/ml青霉素 - 链霉素 加入100毫升10x PBS的890毫升无菌水,并混合
        加入10毫升100x青霉素 - 链霉素股票
        混合和过滤除菌(0.2μm)
        存储在4°C
      6. 25%葡萄糖的1x PBS(50ml)中 将12.5g葡萄糖溶于30ml 1x PBS中
        向50 ml
        中加入1 x PBS 过滤灭菌(0.2μm)
        存储在4°C
      7. DMEM/F12加(25ml) 0.6ml 25%葡萄糖 1.25 ml FBS
        0.25ml 100×青霉素 - 链霉素 0.25ml 100x glutamax
        0.25ml N2补充剂
        22.4ml DMEM/F12
        过滤灭菌(0.2μm)
        存储在4°C
      8. Neurobasal/B27加(50ml) 1.2ml 25%葡萄糖 0.5ml 100x青霉素 - 链霉素 0.5ml 100x glutamax
        1 ml B27补充剂
        47.75 ml neurobasal
        过滤灭菌(0.2μm)
        存储在4°C
      9. 4%多聚甲醛(PFA)在1×PBS中 将4g PFA溶解在装有100ml预热的1×PBS(在65℃)的瓶子中
        加入20μl10 M NaOH,混匀,直至溶液澄清
        用0.45μm过滤器过滤并在4℃保存1周或在-20℃保存几个月。

      致谢

      此协议改编自以前发表的研究,de Diego等人。 (2002),由Bastakis等人进行。 (2015)。这项工作得到了欧盟委员会FP7项目"转换潜力"[合同编号285948],InnovCrete [316223],ARISTEIA I [项目593 MyelinTag]和分子生物学和生物技术研究所的授权。

      参考文献

      1. Bastakis,GG,Savvaki,M.,Stamatakis,A.,Vidaki,M.和Karagogeos,D。(2015)。  对尾骨髓质前脑神经元迁移的多种影响。 129(2):297-306。
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引用:Savvaki, M. and Karagogeos, D. (2016). Olfactory Bulb (OB) Transplants. Bio-protocol 6(17): e1921. DOI: 10.21769/BioProtoc.1921.
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