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In vivo Neurogenesis
体内神经发生   

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Abstract

This protocol shows how to characterize the dynamics of hippocampal neurogenesis in the adult mouse by describing preparation of brain tissue, immunofluorescence of brain sections and confocal stereotactic cell counting.


Materials and Reagents

  1. Mice
  2. 5-Bromo-2′-deoxyuridine (BrdU) (Sigma-Aldrich, catalog number: B5002 )
  3. 0.9% sterile sodium chloride (NaCl) (Fresenius Kabi)
  4. Ketamine hydrochloride (Ketavet, 100 mg/ml) (Pfizer)
  5. Xylazine hydrochloride (Rompun, 20 mg/ml Xylazine) (Bayer)
  6. 4% Paraformaldehyde in phosphate buffer (4% Roti-Histofix) (Roth, catalog number: P087.1 )
  7. Hank’s balanced salt solution (HBSS) (Life Technologies, InvitrogenTM, catalog number: 14170-138 )
  8. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434 )
  9. Sodium phosphate dibasic heptahydrate (Na2HPO4.7H2O) (Sigma-Aldrich, catalog number: S9390 )
  10. Sodium phosphate monobasic monohydrate (NaH2PO4.H2O) (Roth, catalog number: K300.2 )
  11. Potassium chloride (KCl) (AppliChem GmbH, catalog number: A3582 )
  12. Potassium phosphate monobasic (KH2PO4) (GERBU Biotechnik GmbH, catalog number: 2018 )
  13. Hydrochloric acid (HCl, 37%) (Sigma-Aldrich, catalog number: 30721 )
  14. Trizma base (Sigma-Aldrich, catalog number: T1503 )
  15. Horse serum (Biochrom, catalog number: S9135 )
  16. Triton X-100 (Sigma-Aldrich, catalog number: X-100 )
  17. Boric acid (Fluka, catalog number: 15660 )
  18. Sodium tetraborate decahydrate (Sigma-Aldrich, catalog number: S9640 )
  19. Hoechst (33342) (dilute with H2O to 10 mg/ml) (Biotrend, catalog number: 40047 )
  20. Sodium azide (Sigma-Aldrich, catalog number: S2002 )
  21. Superglue
  22. Antibodies (see Table 1 and 2)
  23. Agarose (AppliChem GmbH, catalog number: A8963 )
  24. Phosphate buffered saline (PBS) (20x) (see Recipes)
  25. TBS (10x) (see Recipes)
  26. TBS++ (see Recipes)
  27. Boric buffer (see Recipes)
  28. 0.1 M Phosphate buffer (PB) (see Recipes)
  29. Mowiol (Merck/Calbiochem, catalog number: 475904 ) (see Recipes)

Equipment

  1. Syringe (1 ml syringe 27 G for i.p. injections)
  2. 0.5 ml Eppendorf Safelock tubes
  3. 15 ml and 50 ml Falcon tubes
  4. Petri dish
  5. Micro dissecting scissors
  6. Forceps
  7. Leica VT1200 Vibratome
  8. Brush to transfer slices
  9. Netwell carriers and plates (Corning Incorporated, catalog number: 3477 and 3520 )
  10. Rocking platform
  11. Tube roller mixer
  12. Microscope glass slides
  13. Cover slips
  14. Confocal microscope
  15. Shaker
  16. Centrifuges

Software

  1. ImageJ

Procedure

  1. BrdU administration
    1. Mice are injected intraperitoneal (i.p.) with 300 mg/kg bodyweight BrdU (15 mg/ml diluted in 0.9% NaCl) and perfused after 24 h to analyze proliferation of neural progenitor cells.
    2. Alternatively, to study survival and differentiation of new-born cells mice are injected i.p. on three consecutive days with 300 mg/kg bodyweight per day BrdU and perfused 4 weeks after to analyse BrdU-positive cells.

  2. Transcardial perfusion
    1. Animals are anesthetized with an overdose of Rompun (14 mg/kg bodyweight) and Ketavet (100 mg/kg bodyweight) in 0.9% NaCl.
    2. Mice are transcardially perfused with 30 ml HBSS followed by 10 ml of 4% paraformaldehyde (PFA) dissolved in 0.1 M phosphate buffer pH 7. For transcardial perfusion the thorax cavity is opened, and the right auricle cut with a scissor to allow bleeding. A butterfly cannula is introduced into the left ventricle and mice are perfused with 30 ml HBSS followed by fixation with 10 ml 4% PFA.
    3. Brains are removed and post-fixed overnight in 10 ml 4% PFA in 0.1 M phosphate buffer pH 7 in a 15 ml Falcon tube on a tube roller mixer at 4 °C.
    4. Tissue is washed twice with PBS and may be stored in PBS with 0.01% sodium azide for up to a year.

  3. Vibratome cutting
    1. For coronal vibratome sections (see Figure 1)
      1. The cerebellum is cut, removed.
      2. The brain glued upright with the cutting site using superglue onto the holder plate of the vibratome.
      3. Coronal sections may have a thickness of 50 μm or 100 μm.
        Note: The NeuN antibody does not very well penetrate 100 μm thick sections. If you have to use 100 μm thick sections primary antibody incubation should be 72 h.
    2. For sagittal brain sections (see Figure 1)
      1. Sagittal brain sections are embedded in 2% agarose in PBS.
      2. The brain is then glued in a solid gel block on the lateral side to the holder plate.
      3. Sagittal sections are cut 100 μm thick.
      4. Agarose can be removed from the slices after cutting or may be kept during the staining process in order to stabilize the tissue (especially olfactory bulbs).
    3. Sections are stored in PBS with 0.01% sodium azide at 4 °C. Tissue might be used for up to one year after perfusion.


    Figure 1. Overview of neurogenic niches in coronal and sagittal brain sections (SVZ: subventricular zone; DG: dentate gyrus)

  4. Immunofluorescence staining
    1. For each mouse, 6 coronal brain slices (50 μm thick) 250 μm apart are stained, starting from the first slice, when both, upper and lower blade of the DG, are present on the slice. If saggital sections are used, 3 slices from each hemisphere 3 slices (300 μm) apart are used. Brain sections are placed in net carriers in 12 well plates (2 slices per well) filled with 4 ml 0.1 M Tris buffer (pH 7.4) supplemented with 0.8% NaCl (TBS). The sections are washed three times in TBS each 15 min at RT on a rocking platform (50 rpm).
    2. BrdU staining
      1. BrdU staining requires DNA denaturization by incubating the brain sections in 2 N HCl (always prepare fresh) at 37 °C on a shaker for 30 min.
      2. Thereafter, sections are neutralized by washing in Boric buffer for 10 min on a shaker at RT.
      3. Next, sections are washed six times in TBS for 15 min each at RT.
    3. Blocking of unspecific antibody binding is performed by incubating sections for 1 h in TBS++ in net carriers at RT.
    4. Sections are transferred to 0.5 ml Eppendorf Safelock tubes (2 sections per tube) containing 200 μl TBS++ and the diluted primary antibodies, and incubated at 4 °C for 24-72 h. For this 12 tubes are put in a 50 ml Falcon and rotated at 4 °C on a tube roller mixer. A selection of primary antibodies is listed in Table 1.

      Table 1. Primary Antibody list
      Antibody
      Host species
      Manufacturer
      Catalog number
      Dilution
      anti-BrdU
      rat
      AbD Serotec
      OBT0030CX
      1:500
      anti-cleaved Caspase-3
      rabbit
      Cell Signaling
      9661
      1:200
      anti-DCX (C-18)
      goat
      Santa Cruz
      sc-8066
      1:250
      anti-GFAP
      mouse
      Millipore
      MAB360
      1:400
      anti-GFAP
      rabbit
      Millipore
      AB5804
      1:400
      anti-GFP
      chicken
      Aves
      GFP-1020
      1:1,000
      anti-NeuN
      mouse
      Millipore
      MAB377
      1:200
      anti-S100β
      mouse
      Sigma-Aldrich
      S2532
      1:200
      anti-Sox2
      rabbit
      Abcam
      ab92494
      1:500
      anti-Tbr2 (Eomes)
      rabbit
      Abcam
      ab23345
      1:500
      Tbr2 Antibody: Some lots work for paraffin sections, others work for the vibratome sections that are used here (see Figure 2). Ask Abcam for information about that or order 2-3 vials to test different lots.

    5. After incubation sections are transferred back to net carriers in 12 well plates, washed three times with TBS at RT.
    6. After blocking in TBS++ for 30 min at RT sections are transferred again into 0.5 ml Eppendorf Safelock tubes containing the diluted secondary antibody mix in TBS++. Sections in Eppdorf tubes in Falcons are incubated in secondary antibodies at 4 °C on a tube roller mixer for 2 h. Secondary antibodies are listed in Table 2. Hoechst 33342 (1:10,000) is used to counterstain DNA and added to the secondary antibody mix.

      Table 2. Secondary Antibody list
      Antibody
      Manufacturer
      Catalog number
      Dilution
      donkey anti-chicken DyLight488
      Dianova
      703-485-155
      1:400
      donkey anti-goat Alexa 488
      Invitrogen
      A-11055
      1:400
      donkey anti-goat Alexa 456
      Invitrogen
      A11057
      1:400
      donkey anti-goat Alexa 647
      Dianova
      705-605-147
      1:400
      donkey anti-mouse DyLight488
      Dianova
      715-485-150
      1:400
      donkey anti-mouse Alexa 546
      Invitrogen
      A10036
      1:400
      donkey anti-rabbit DyLight488
      Dianova
      711-485-152
      1:400
      donkey anti-rabbit DyLight 649
      Dianova
      711-495-152
      1:400
      donkey anti-rat Alexa 488
      Dianova
      712-545-150
      1:400
      donkey anti-rat rhodamine red
      Dianova
      712-296-150
      1:400

    7. Finally sections are placed back into net carriers in 12 well plates washed three times for 15 min with TBS and additionally 4 times for 1 min in TBS at RT.
    8. Sections are floated in 0.1 M PB in a Petri dish, mounted on glass slides and embedded with 100 μl Mowiol.

  5. Confocal microscopy
    1. Confocal microscope pictures can be taken with a 20x, 40x or 60x objective on a confocal microscope.
    2. Cell numbers can be counted manually or by using the Cell counter plug in of ImageJ.
    3. Cell numbers are normalized to the volume of the DG granule cell layer measured by ImageJ.

Representative data

Not all Tbr2 Eomes antibody (AB) lots work for this protocol. Below are representative pictures and sample lots that show a specific staining (A) of Tbr2 in vibratome sections and antibody lots that give only unspecific background staining (B). Red arrows in A show Tbr2 staining. The green arrows indicate unspecific background staining of glia-like cells that might appear in the specific antibody lots. This background staining is however very well distinguishable from the nuclear Tbr2 staining.


Figure 2. A specific staining (A) of Tbr2 in vibratome sections and antibody lots that give only unspecific background staining (B).

Recipes

  1. PBS (20x)
    NaCl
    160 g/L
    Na2HPO4
    23 g/L
    Na2HPO4
    28.84 g/L
    KCl
    4 g/L
    KH2PO4
    4 g/L
    Adjust pH to 7.4 with HCl and fill volume up to 1 L with dH2O.
  2. TBS (10x)
    Trizma base
    24.23 g/L
    NaCl
    80.06 g/L
    Mix in 800 ml ultra-pure water, adjust pH to 7.6 with pure HCl and fill up to 1 L.
  3. TBS ++
    TBS
    100 ml
    Horse serum
    3 ml
    Triton X-100
    0.25 ml
  4. Boric buffer
    Boric acid
    3.1 g/L
    Sodium tetraborate
    4.75 g/L
    Mix in 800 ml ultra pure water, adjust pH to 7.6 and fill up to 1 L.
  5. 0.1 M Phosphate buffer
    0.2 M Monobasic Stock
    NaH2PO4.H2O
    13.9 g/500 ml
    0.2 M Dibasic stock

    Na2HPO4.7H2O
    53.65 g/L
    Combine indicated amounts of 0.2 M monobasic and 0.2 M dibasic stock solutions and bring volume up to 600 ml.
    0.2 M Monobasic Stock
    0.2 M Dibasic Stock
    pH
    57 ml
    243 ml
    7.4
  6. Mowiol
    1x PBS
    40 ml

    Mowiol
    10 g
    → stir for 24 h
    Add Glycerol
    20 ml
    → stir for 24 h
    Centrifuge 15 min at 5,000 rpm, RT
    Aliquot and store at -20 °C

Acknowledgments

This protocol is adapted from Seib et al. (2012).

References

  1. Seib, D. R., Corsini, N. S., Ellwanger, K., Plaas, C., Mateos, A., Pitzer, C., Niehrs, C., Celikel, T.nd Martin-Villalba, A. (2013). Loss of Dickkopf-1 restores neurogenesis in old age and counteracts cognitive decline. Cell Stem Cell 12(2): 204-214.

简介

该协议显示如何描述成年小鼠海马神经发生的动力学描述大脑组织的准备,脑切片的免疫荧光和共聚焦立体定向细胞计数。


材料和试剂

  1. 小鼠
  2. 5-溴-2'-脱氧尿苷(BrdU)(Sigma-Aldrich,目录号:B5002)
  3. 0.9%无菌氯化钠(NaCl)(Fresenius Kabi)
  4. 盐酸氯胺酮(Ketavet,100mg/ml)(Pfizer)
  5. 甲苯噻嗪盐酸盐(Rompun,20mg/ml甲苯噻嗪)(Bayer)
  6. 4%多聚甲醛的磷酸盐缓冲液(4%Roti-Histofix)(Roth,目录号:P087.1)
  7. Hank's平衡盐溶液(HBSS)(Life Technologies,Invitrogen TM ,目录号:14170-138)
  8. 氯化钠(NaCl)(Sigma-Aldrich,目录号:31434)
  9. 将磷酸氢二钠七水合物(Na 2 HPO 4+),7H 2 O(Sigma-Aldrich,目录号: S9390)
  10. 磷酸二氢钠一水合物(NaH 2 PO 4 PO 4,H 2 O 2)(Roth,目录号:K300。 2)
  11. 氯化钾(KCl)(AppliChem GmbH,目录号:A3582)
  12. 磷酸二氢钾(KH 2 PO 4)(GERBU Biotechnik GmbH,目录号:2018)
  13. 盐酸(HCl,37%)(Sigma-Aldrich,目录号:30721)
  14. Trizma碱(Sigma-Aldrich,目录号:T1503)
  15. 马血清(Biochrom,目录号:S9135)
  16. Triton X-100(Sigma-Aldrich,目录号:X-100)
  17. 硼酸(Fluka,目录号:15660)
  18. 十水合四硼酸钠(Sigma-Aldrich,目录号:S9640)
  19. Hoechst(33342)(用H 2 O稀释至10mg/ml)(Biotrend,目录号:40047)
  20. 叠氮化钠(Sigma-Aldrich,目录号:S2002)
  21. 超能量
  22. 抗体(见表1和2)
  23. 琼脂糖(AppliChem GmbH,目录号:A8963)
  24. 磷酸盐缓冲盐水(PBS)(20x)(参见配方)
  25. TBS(10x)(参见配方)
  26. TBS ++(参见配方)
  27. 硼缓冲液(见配方)
  28. 0.1 M磷酸盐缓冲液(PB)(参见配方)
  29. Mowiol(Merck/Calbiochem,目录号:475904)(参见Recipes)

设备

  1. 注射器(1ml注射器,27G,用于腹膜内注射)
  2. 0.5 ml Eppendorf Safelock管
  3. 15 ml和50 ml Falcon管
  4. 培养皿
  5. 微解剖剪刀
  6. 镊子
  7. Leica VT1200 Vibratome
  8. 刷子传输切片
  9. 网络载体和板(Corning Incorporated,目录号:3477和3520)
  10. 摇台
  11. 管滚子搅拌器
  12. 显微镜玻片
  13. 盖玻片
  14. 共焦显微镜
  15. 振动器
  16. 离心机

软件

  1. ImageJ

程序

  1. BrdU管理
    1. 小鼠腹膜内(i.p.)注射300mg/kg体重的BrdU(15mg/ml,稀释在0.9%NaCl中),24小时后灌注以分析神经祖细胞的增殖。
    2. 或者,为了研究新生动物细胞的存活和分化,将小鼠腹腔注射。 连续三天,每天用300mg/kg体重的BrdU并在4周后灌注分析BrdU阳性细胞。

  2. 经心灌注
    1. 在0.9%NaCl中用过量的Rompun(14mg/kg体重)和Ketavet(100mg/kg体重)麻醉动物。
    2. 小鼠用30ml HBSS,然后用溶解在pH7的0.1M磷酸盐缓冲液中的10ml 4%多聚甲醛(PFA)经心脏灌注。对于经心脏灌注,打开胸腔,用剪刀切割右心耳以允许出血。 将蝴蝶插管引入左心室,用30ml HBSS灌注小鼠,然后用10ml 4%PFA固定。
    3. 去除脑,并在4ml的管式滚动混合器中在15ml Falcon管中在10ml的4%PFA的0.1M磷酸盐缓冲液pH7中后固定过夜。
    4. 组织用PBS洗涤两次,并可以在含有0.01%叠氮化钠的PBS中储存长达一年
  3. 振动切削
    1. 对于冠状振动器部分(参见图1)
      1. 小脑被切除,移除。
      2. 大脑使用超强力胶与切割部位垂直胶合到振动器的固定板上。
      3. 冠状截面可以具有50μm或100μm的厚度 注意:NeuN抗体不能很好地穿透100μm厚的切片。 如果你必须使用100μm厚的切片,一抗孵育应该是72小时。
    2. 对于矢状脑切片(参见图1)
      1. 将矢状脑切片包埋在PBS中的2%琼脂糖中。
      2. 然后将大脑粘在固定的凝胶块中,固定在固定板的侧面
      3. 矢状切面切成100μm厚。
      4. 琼脂糖可以在切割后从切片中除去,或者可以在染色过程中保持,以稳定组织(特别是嗅球)。
    3. 切片在4℃下储存在含有0.01%叠氮化钠的PBS中。 组织可能在灌注后使用长达一年。


    图1.冠状和矢状脑切片(SVZ:室下区; DG:齿状回)中的神经源性龛的概述

  4. 免疫荧光染色
    1. 对于每只小鼠,当DG的上刀片和下刀片都存在于切片上时,从第一切片开始,对相隔250μm的6个冠状脑切片(50μm厚)进行染色。 如果使用矢状截面,则使用来自每个半球3个切片(300μm)的3个切片。 将脑切片置于填充有补充有0.8%NaCl(TBS)的4ml 0.1M Tris缓冲液(pH 7.4)的12孔板(每孔2个切片)中的净载体中。 将切片在室温下在摇动平台(50rpm)上在TBS中每15分钟洗涤三次。
    2. BrdU染色
      1. BrdU染色需要DNA变性,通过将脑切片在37℃下在振荡器上在2N HCl(总是新鲜制备)中温育30分钟。
      2. 此后,通过在室温下在摇床上在硼缓冲液中洗涤10分钟来中和切片。
      3. 接下来,将切片在室温下在TBS中洗涤6次,每次15分钟。
    3. 通过在室温下在TBS ++中在净载体中孵育切片1小时来进行非特异性抗体结合的阻断。
    4. 将切片转移至含有200μlTBS ++和稀释的一抗的0.5ml Eppendorf Safelock管(每管2个切片),并在4℃下孵育24-72小时。 为此,将12个管放入50ml Falcon中并在管式滚筒混合器上在4℃下旋转。 表1列出了一级抗体的选择
      表1.一级抗体列表
      抗体
      主机物种
      制造商
      目录号
      稀释
      抗BrdU
      大鼠
      AbD Serotec
      OBT0030CX
      1:500
      抗裂解的Caspase-3

      单元格信号
      9661
      1:200
      抗DCX(C-18)
      山羊
      圣克鲁斯
      sc-8066
      1:250
      抗GFAP
      小鼠
      Millipore
      MAB360
      1:400
      抗GFAP

      Millipore
      AB5804
      1:400
      抗GFP

      Aves
      GFP-1020
      1:1,000
      抗NeuN
      小鼠
      Millipore
      MAB377
      1:200
      抗S100β
      小鼠
      Sigma-Aldrich
      S2532
      1:200
      反Sox2

      Abcam
      ab92494
      1:500
      抗Tbr2(Eomes)

      Abcam
      ab23345
      1:500
      Tbr2抗体:一些批次用于石蜡切片,另一些用于此处使用的振动切片(参见图2)。问问Abcam有关信息或订购2-3瓶以测试不同的批次
    5. 孵育后,将切片转移回12孔板中的网状载体,在室温下用TBS洗涤三次
    6. 在TBS ++中在室温下封闭30分钟后,将切片再次转移到含有在TBS ++中的稀释的二级抗体混合物的0.5ml Eppendorf Safelock管中。将来自Falcons的Eppdorf管中的切片在2℃下在管式滚筒混合器上在二抗中孵育2小时。第二抗体列于表2中。使用Hoechst 33342(1:10,000)复染DNA并加入第二抗体混合物中。

      表2.二级抗体列表
      抗体
      制造商
      目录号
      稀释
      驴反鸡DyLight488
      Dianova

      1:400
      驴抗山羊Alexa 488
      Invitrogen
      A-11055
      1:400
      驴抗山羊Alexa 456
      Invitrogen
      A11057
      1:400
      驴抗山羊Alexa 647
      Dianova

      1:400
      驴抗小鼠DyLight488
      Dianova
      715-485-150
      1:400
      驴抗小鼠Alexa 546
      Invitrogen
      A10036
      1:400
      驴抗兔DyLight488
      Dianova
      711-485-152
      1:400
      驴抗兔DyLight 649
      Dianova
      711-495-152
      1:400
      驴抗大鼠Alexa 488
      Dianova
      712-545-150
      1:400
      驴抗大鼠罗丹明红
      Dianova
      712-296-150
      1:400
      1:400
      驴抗大鼠罗丹明红
      Dianova
      712-296-150
      1:400
      ...
    7. 将细胞数标准化为通过ImageJ测量的DG颗粒细胞层的体积

代表数据

不是所有Tbr2 Eomes抗体(AB)批次都适用于此协议。 下面是代表性的图片和样品批次,其显示了仅在非特异性背景染色(B)的vibratome切片和抗体批次中Tbr2的特异性染色(A)。 A中的红色箭头显示Tbr2染色。 绿色箭头表示可能出现在特异性抗体批次中的胶质样细胞的非特异性背景染色。 然而,这种背景染色与核Tbr2染色非常相似

图2.在只有非特异性背景染色(B)的vibratome切片和抗体批次中Tbr2的特异性染色(A)。

食谱

  1. PBS(20x)
    NaCl
    160 g/L
    Na HPO 4
    23克/升
    Na HPO 4
    28.84克/升
    KCl
    4 g/L
    KH 2 PO 4
    4 g/L
    用HCl调节pH至7.4,用dH 2 O填充体积至1L
  2. TBS(10x)
    Trizma基地
    24.23克/升
    NaCl
    80.06 g/L
    混合在800毫升超纯水中,用纯HCl调节pH至7.6,并填充至1升
  3. TBS ++
    TBS
    100 ml
    马血清
    3 ml
    Triton X-100
    0.25 ml
  4. 硼酸缓冲液
    硼酸
    3.1 g/L
    四硼酸钠
    4.75g/L
    在800ml超纯水中混合,调节pH至7.6并填充至1L。
  5. 0.1 M磷酸盐缓冲液
    0.2 M一次性库存
    NaH <2> PO 4 H O
    13.9g/500ml
    0.2 M二碱性股票

    lt; sub> 4< sub> 7H O
    53.65克/升
    合并所示量的0.2M一元和0.2M二元储备溶液,并使体积达到600ml。
    0.2 M一元股票
    0.2 M二碱基料
    pH
    57 ml
    243 ml
    7.4
  6. Mowiol
    1x PBS
    40 ml

    Mowiol
    10克
    →搅拌24 h
    添加甘油
    20ml
    →搅拌24小时
    在5,000rpm,RT下离心15分钟
    等分并存储在-20°C

致谢

该协议改编自Seib等人(2012)。

参考文献

  1. Seib,DR,Corsini,NS,Ellwanger,K.,Plaas,C.,Mateos,A.,Pitzer,C.,Niehrs,C.,Celikel,T.nd Martin-Villalba, target ="_ blank"href ="http://www.ncbi.nlm.nih.gov/pubmed/23395445"> Dickkopf-1的缺失恢复了老年人的神经发生并抵消了认知衰退。细胞 干细胞 12(2):204-214
  • English
  • 中文翻译
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Seib, D. R. and Martin-Villalba, A. (2013). In vivo Neurogenesis. Bio-protocol 3(15): e841. DOI: 10.21769/BioProtoc.841.
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