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Neuronal Morphology Analysis
神经元形态分析   

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参见作者原研究论文

本实验方案简略版
Cell Stem Cell
Feb 2013

Abstract

This protocol describes how to visualize neuronal morphology and how to determine neuronal complexity of immature and mature hippocampal neurons in the mouse in vivo including tissue preparation, staining of brain sections and confocal cell analysis.

Materials and Reagents

  1. Mice
  2. 0.9% sterile sodium chloride (NaCl) (Fresenius Kabi)
  3. Ketamine hydrochloride (Ketavet, 100 mg/ml) (Pfizer)
  4. Xylazine hydrochloride (Rompun, 20 mg/ml Xylazine) (Bayer)
  5. 4% Paraformaldehyde in phosphate buffer (4% Roti-Histofix) (Roth, catalog number: P087.1 )
  6. Hank’s balanced salt solution (HBSS) (Life Technologies, InvitrogenTM, catalog number: 14170-138 )
  7. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434 )
  8. Sodium phosphate dibasic heptahydrate (Na2HPO4.7H2O) (Sigma-Aldrich, catalog number: S9390 )
  9. Sodium phosphate monobasic monohydrate (NaH2PO4.H2O) (Roth, catalog number: K300.2 )
  10. Potassium chloride (KCl) (AppliChem GmbH, catalog number: A3582 )
  11. Potassium phosphate monobasic (KH2PO4) (Gerbu, catalog number: 2018 )
  12. Sodium azide (Sigma-Aldrich, catalog number: S2002 )
  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. Chicken anti-GFP antibody (Aves, catalog number: GFP-1020 )
  18. Goat anti-Doublecortin antibody (DCX, C18) (Santa Cruz, catalog number: sc-8066 )
  19. Mouse anti-NeuN antibody (EMD Millipore, catalog number: MAB377 )
  20. Donkey anti-chicken DyLight488 antibody (Dianova, catalog number: 703-485-155 )
  21. Donkey anti-goat Alexa 647 antibody (Dianova, catalog number: 705-605-147 )
  22. Donkey anti-mouse Alexa 546 antibody (Life Technologies, InvitrogenTM, catalog number: A10036 )
  23. Hoechst (33342) (Biotrend, catalog number: 40047 )
  24. Gelatine to coat glass slides (Sigma-Aldrich, catalog number: G7041 )
  25. Chromium (III) potassium sulfate dodecahydrate (Sigma-Aldrich, catalog number: 60152 )
  26. Bromothymol Blue sodium salt (Sigma-Aldrich, catalog number: 114421 )
  27. FD Rapid GolgiStainTM Kit (FD NeuroTechnologies, catalog number: PK401 )
  28. Millipore water
  29. Ethanol (Sigma-Aldrich, catalog number: 459844 )
  30. Xylene (Sigma-Aldrich, catalog number: 33817 )
  31. Eukitt (Fluka, catalog number: 03989 )
  32. Agarose (AppliChem GmbH, catalog number: A8963 )
  33. Phosphate buffer saline (PBS) (20x) (see Recipes)
  34. TBS (10x) (see Recipes)
  35. TBS++ (see Recipes)
  36. 0.1 M Phosphate buffer (see Recipes)
  37. Gelatine to coat glass slides (see Recipes)
  38. 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. Micro dissecting scissors
  5. Forceps
  6. Leica VT1200 Vibratome
  7. Brush to transfer slices
  8. Netwell carriers and plates (Corning Inc., catalog numbers: 3477 and 3520 )
  9. Rocking platform
  10. Tube roller mixer
  11. Hot plate stirrer
  12. Staining containers
  13. Microscope glass slides
  14. Cover slips
  15. Confocal microscope
  16. Centrifuge

Software

  1. Amira Filament Editor Analysis (Visage Imaging) or other neuron morphology analysis software

Procedure

  1. Tissue preparation
    1. Transcardial perfusion
      Animals are anesthetized with an overdose of Rompun (14 mg/kg bodyweight) and Ketavet (100 mg/kg bodyweight) in 0.9% NaCl.
      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 in the left ventricle and mice are perfused with 30 ml HBSS followed by fixation with 10 ml 4% PFA.
      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.
      Tissue is washed twice with PBS and may be stored in PBS with 0.01% sodium azide for up to a year.

  2. Neuronal morphology analysis of immature dentate gyrus neurons
    1. Vibratome cutting
      1. For coronal vibratome sections (see Figure 1), the cerebellum is cut, removed and the brain glued upright with the cutting site using superglue onto the holder plate of the vibratome. 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 sections (see Figure 1) brains are embedded in 2% agarose in PBS. The brain is then glued in a solid gel block on the lateral side to the holder plate. Sagittal sections are cut 100 μm thick. Agarose can be removed from the slices during cutting or may be kept during the staining process in order to stabilize the tissue (especially olfactory bulbs).
      3. Sections can be 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).

    1. Immunofluorescence staining
      1. For each mouse, 4 brain slices (50 or 100 μm thick, 250 μm or 300 μm apart, respectively) are stained. 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 8% NaCl (TBS). The sections are washed three times in TBS each 15 min at RT on a rocking platform (50 rpm).
      2. Blocking of unspecific antibody binding is performed by incubating sections for 1 h in TBS++ at RT.
      3. 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.
        1. For staining of GFP/YFP of either genetically or retrovirally labeled immature neurons sections are stained with the primary chicken anti-GFP antibody (1:1,000).
        2. Otherwise non-labeled immature neurons are stained with the goat anti-Doublecortin antibody (1:200). Mouse anti-NeuN (1:200) might be used as additional marker to determine cell maturity.
      4. After incubation sections are transferred back to net carriers in 12 well plates, washed three times with TBS at RT.
      5. 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.
        1. Secondary antibodies are diluted 1:400: Donkey anti-chicken DyLight488, donkey anti-goat Alexa 647 or donkey anti-mouse Alexa 546.
        2. Hoechst 33342 (1:10,000) is used to counterstain DNA and added to the secondary antibody mix.
      6. 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.
      7. Sections are floated in 0.1 M PB in a Petri dish, mounted on glass slides and embedded with 100 μl Mowiol.
    2. Confocal microscope pictures are taken with a 40x objective on a confocal microscope. Branching points and total dendrite length are measured using Amira Filament Editor Analysis (Visage Imaging).

  3. Neuronal morphology analysis of mature hippocampal neurons
    1. For analysis of neuronal morphology of mature CA or DG neurons PFA fixed brains are cut in two hemispheres and stained with the FD Rapid GolgiStainTM Kit.
      1. Hemispheres are incubated in impregnation solution (A and B) for 2 weeks at RT in the dark.
      2. After that tissue is transferred into solution C and stored for five days at 4 °C protected from light.
      3. The tissue is cut in 100 μm thick coronal sections floating in solution C with a Leica VT1200 vibratome and mounted on gelatine coated glass slides. To coat glass slides 1.5 g gelatine and 0.25 g chromium potassium sulfate are mixed with 500 ml distilled water, a few crystals of bromthymol blue are added as preservative, heated up to 60 °C in order to dissolve gelatine and then glass slides are dipped into the solution and the lower part of the slide is cleaned with a tissue. Coated slides are dried overnight at RT.
      4. Mounted slides are washed 2x for 2 min in Millipore water.
      5. Sections are stained for 10 min in staining solution (40 ml solution D, 40 ml solution E and 80 ml Millipore water) at RT.
      6. Sections are washed 2 x 4 min in Millipore water, once for 4 min in 50% ethanol (EtOH), once for 4 min in 75% EtOH, once for 4 min in 95% EtOH and four times for 4 min in 100% EtOH at RT.
      7. Finally slices are washed three times for 4 min in xylene and embedded with Eukitt.
    2. Neurons of the dentate gyrus and CA regions can be analysed with this method. Stacks can be recorded on a confocal microscope with a 40x objective. Branching points and total dendrite length are measured using Amira Filament Editor Analysis (Visage Imaging) (see Figure 2).

Representative data


Figure 2. Light microscope (A) and confocal microscope picture (B) of Golgi stained neurons. Picture shown in B (left panel) was used for image analysis with Amira (right panel).

Recipes

  1. PBS (20x)
    NaCl
    160 g/L
    Na2HPO4
    23 g/L
    NaH2PO4
    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. 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
  5. Gelatine to coat glass slides
    Gelatine
    1.5 g
    Chromium (III) potassium sulfate dodecahydrate
    0.25 g
    Add a few crystals of bromthymol blue as a preservative
    Fill up to 500 ml with H2O and heat up to 60 °C to dissolve gelatin.
  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. and 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

该协议描述了如何可视化神经元形态和如何确定小鼠体内未成熟和成熟海马神经元的神经元复杂性,包括组织制备,脑切片染色和共聚焦细胞分析。

材料和试剂

  1. 小鼠
  2. 0.9%无菌氯化钠(NaCl)(Fresenius Kabi)
  3. 盐酸氯胺酮(Ketavet,100mg/ml)(Pfizer)
  4. 甲苯噻嗪盐酸盐(Rompun,20mg/ml甲苯噻嗪)(Bayer)
  5. 4%多聚甲醛的磷酸盐缓冲液(4%Roti-Histofix)(Roth,目录号:P087.1)
  6. Hank's平衡盐溶液(HBSS)(Life Technologies,Invitrogen TM ,目录号:14170-138)
  7. 氯化钠(NaCl)(Sigma-Aldrich,目录号:31434)
  8. 将磷酸氢二钠七水合物(Na 2 HPO 4+),7H 2 O(Sigma-Aldrich,目录号: S9390)
  9. 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)
  10. 氯化钾(KCl)(AppliChem GmbH,目录号:A3582)
  11. 磷酸二氢钾(KH 2 PO 4 sub)(Gerbu,目录号:2018)
  12. 叠氮化钠(Sigma-Aldrich,目录号:S2002)
  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. 鸡抗GFP抗体(Aves,目录号:GFP-1020)
  18. 山羊抗双皮质蛋白抗体(DCX,C18)(Santa Cruz,目录号:sc-8066)
  19. 小鼠抗NeuN抗体(EMD Millipore,目录号:MAB377)
  20. 驴抗鸡DyLight488抗体(Dianova,目录号:703-485-155)
  21. 驴抗山羊Alexa 647抗体(Dianova,目录号:705-605-147)
  22. 驴抗小鼠Alexa 546抗体(Life Technologies,Invitrogen TM ,目录号:A10036)
  23. Hoechst(33342)(Biotrend,目录号:40047)
  24. 明胶涂覆载玻片(Sigma-Aldrich,目录号:G7041)
  25. 铬(III)硫酸钾十二水合物(Sigma-Aldrich,目录号:60152)
  26. 溴百里酚蓝钠盐(Sigma-Aldrich,目录号:114421)
  27. FD Rapid GolgiStain TM Kit(FD NeuroTechnologies,目录号:PK401)
  28. 微孔水
  29. 乙醇(Sigma-Aldrich,目录号:459844)
  30. 二甲苯(Sigma-Aldrich,目录号:33817)
  31. Eukitt(Fluka,目录号:03989)
  32. 琼脂糖(AppliChem GmbH,目录号:A8963)
  33. 磷酸盐缓冲盐水(PBS)(20x)(见配方)
  34. TBS(10x)(参见配方)
  35. TBS ++(参见配方)
  36. 0.1 M磷酸盐缓冲液(参见配方)
  37. 明胶涂层玻璃载玻片(见配方)
  38. Mowiol(Merck/Calbiochem,目录号:475904)(参见配方)

设备

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

软件

  1. Amira细丝编辑分析(Visage Imaging)或其他神经元形态分析软件

程序

  1. 组织制备
    1. 经心脏灌注
      在0.9%NaCl中用过量的Rompun(14mg/kg体重)和Ketavet(100mg/kg体重)麻醉动物。 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。在左心室中引入蝶形插管,用30ml HBSS灌注小鼠,然后用10ml 4%PFA固定。
      去除脑,并在4ml的管式滚动混合器中的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。
      组织用PBS洗涤两次,并且可以在具有0.01%叠氮化钠的PBS中储存长达一年。

  2. 未成熟齿状回神经元的神经元形态分析
    1. 振动切削
      1. 对于冠状vibratome切片(见图1),小脑被切除,删除,大脑与切割位置使用superglue直接粘贴到vibratome的固定板上。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. 对于矢状切片(参见图1),将脑包埋在PBS中的2%琼脂糖中。 然后将脑在固定的凝胶块中胶合到保持器板的侧面上。 矢状切面切成100μm厚。 琼脂糖可以在切割期间从切片中移除,或者可以在染色过程期间保持,以稳定组织(特别是嗅球)。
      3. 切片可以在含有0.01%叠氮化钠的PBS中在4℃下保存。 组织可能在灌注后使用长达一年。

    图1.共同 颈椎和矢状脑切面(SVZ:室下区; DG:齿状回)的神经源性龛位概述。

    1. 免疫荧光染色
      1. 对于每只小鼠,染色4个脑切片(50或100μm厚,分别为250μm或300μm)。将脑切片置于装有补充有8%NaCl(TBS)的4ml 0.1M Tris缓冲液pH7.4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. 通过在RT下在TBS ++中孵育切片1小时进行非特异性抗体结合的阻断
      3. 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。
        1. 对于基因或反转录病毒标记的未成熟神经元的GFP/YFP染色,切片用原代鸡抗GFP抗体(1:1,000)染色。
        2. 否则,未标记的未成熟神经元用山羊抗双皮质素抗体(1:200)染色。可以使用小鼠抗NeuN(1:200)作为额外的标记物来确定细胞成熟度
      4. 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
      5. 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。
        1. 二抗用1:400稀释:驴抗鸡DyLight488,驴抗山羊Alexa 647或驴抗小鼠Alexa 546.
        2. Hoechst 33342(1:10,000)用于复染DNA并加入第二抗体混合物中。
      6. 最后,将切片置于12孔板中的净载体中,用TBS洗涤三次,15分钟,在室温下,在TBS中洗涤另外4次,每次1分钟。
      7. 将切片漂浮在培养皿中的0.1M PB中,固定在载玻片上并用100μlMowiol包埋。
    2. 用共聚焦显微镜上的40x物镜拍摄共聚焦显微镜照片。 使用Amira Filament Editor Analysis(Visage Imaging)测量分枝点和总枝晶长度
  3. 成熟海马神经元的神经元形态分析
    1. 为了分析成熟CA或DG神经元的神经元形态,将PFA固定的脑切成两个半球,并用FD Rapid GolgiStain TM试剂盒染色。
      1. 将半球在浸渍溶液(A和B)中在黑暗中室温下孵育2周
      2. 之后,将组织转移到溶液C中,并在4℃避光保存5天
      3. 使用Leica VT1200振动台将组织切割成在溶液C中漂浮的100μm厚的冠状切片,并且安装在涂覆有明胶的载玻片上。为了涂覆载玻片,将1.5g明胶和0.25g硫酸铬钾与500ml蒸馏水混合,加入几种溴百里酚蓝的晶体作为防腐剂,加热至60℃以溶解明胶,然后将载玻片浸入用组织清洁溶液和载玻片的下部。包被的载玻片在RT下干燥过夜
      4. 将装载的载玻片在Millipore水中洗涤2次,每次2分钟。
      5. 切片在室温下在染色溶液(40ml溶液D,40ml溶液E和80ml Millipore水)中染色10分钟。
      6. 将切片在Millipore水中洗涤2×4分钟,在50%乙醇(EtOH)中洗涤4分钟,在75%EtOH中洗涤4分钟,在95%EtOH中洗涤4分钟,在100%EtOH中洗涤4分钟4次在 RT。
      7. 最后,将切片在二甲苯中洗涤3次,每次4分钟,并用Eukitt包埋。
    2. 齿状回和CA区的神经元可以用这种方法分析。 可以在具有40x物镜的共焦显微镜上记录堆。 使用Amira Filament Editor Analysis(Visage Imaging)(参见图2)测量分枝点和总枝晶长度。

代表数据


图2.高尔基体染色神经元的光显微镜(A)和共焦显微镜图片(B)。使用B(左图)所示的图像用于Amira(右图)的图像分析。

食谱

  1. PBS(20x)
    NaCl
    160 g/L
    Na HPO 4
    23克/升
    NaH 2 PO 4 sub
    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. 0.1 M磷酸盐缓冲液
    0.2 M一元股票
    NaH 2 2 PO 4 4 H <2> O
    13.9g/500ml
    0.2 M二碱基料
    lt; sub> 4< sub> 7H O
    53.65克/升
    合并指示量的0.2M一元和0.2M二元储备溶液,并使体积达到600毫升
    0.2 M一元股票
    0.2 M二碱基料
    pH
    57 ml
    243 ml
    7.4
  5. 明胶涂布玻璃载玻片
    明胶
    1.5 g
    铬(III)硫酸钾十二水合物
    0.25 g
    加入几种溴百里酚蓝色结晶作为防腐剂 用H 2 O填充至500ml,加热至60℃以溶解明胶。
  6. Mowiol
    1x PBS
    40 ml

    Mowiol
    10克
    →搅拌24小时
    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,D.R.,Corsini,N.S。,Ellwanger,K.,Plaas,C.,Mateos,A.,Pitzer,C.,Niehrs,C.,Celikel,T.and Martin-Villalba, Dickkopf-1的缺失可恢复老年人的神经发生并抵消认知衰退。 Cell Stem Cell 12(2):204-214。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Seib, D. R. and Martin-Villalba, A. (2013). Neuronal Morphology Analysis. Bio-protocol 3(15): e842. DOI: 10.21769/BioProtoc.842.
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