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In these culture models, the normal cytoarchitecture and local neuronal circuits of the spinal cord are preserved, offering a compromise between dissociated cell cultures and complete animal studies. The addition of 5-ethynyl-2’-deoxyuridine (EdU) to the culture medium allows for the detection of proliferating cells.

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Organotypic Spinal Cord Slice Cultures and a Method to Detect Cell Proliferation in These Slices
器官型脊髓片培养和脊髓片上细胞增殖的检测方法

神经科学 > 细胞机理 > 细胞分离和培养
作者: Jillian M. Daniel
Jillian M. DanielAffiliation: School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom
Bio-protocol author page: a3566
Jim Deuchars
Jim DeucharsAffiliation: School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom
Bio-protocol author page: a3567
 and Susan A. Deuchars
Susan A. DeucharsAffiliation: School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom
For correspondence: s.a.deuchars@leeds.ac.uk
Bio-protocol author page: a3568
Vol 6, Iss 19, 10/5/2016, 1150 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1951

[Abstract] In these culture models, the normal cytoarchitecture and local neuronal circuits of the spinal cord are preserved, offering a compromise between dissociated cell cultures and complete animal studies. The addition of 5-ethynyl-2’-deoxyuridine (EdU) to the culture medium allows for the detection of proliferating cells.
Keywords: Neurogenesis(神经发生), Spinal cord dissection(脊髓的解剖), Ependymal cell(室管膜细胞), 5-ethynyl-2'-deoxyuridine(5-ethynyl-2’-脱氧尿苷)

[Abstract]

Materials and Reagents

  1. Filter paper, 55 mm (Sigma-Aldrich, Whatman®, catalog number: WHA1441055 )
  2. Millicell organotypic filter inserts 0.4 µm, 30 mm (EDM Millipore, catalog number: Picmorg50 )
  3. 6-well culture dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140675 )
  4. 24-well histology trays (Sigma-Aldrich, CELLSTAR®, catalog number: 662160 )
  5. 50 mm sterilin Petri dish (Camlab, catalog number: p17118 )
    Note: This link leads to a range of Petri dishes.
  6. Pipette tips, 1,000 µl (SARSTEDT, catalog number: 70.762.010 )
  7. Pipette tips, 200 µl (SARSTEDT, catalog number: 70.760.012 )
  8. Syringe
  9. Glass microscope slides (Academy Science, catalog number: N/A143 )
  10. Glass coverslips (VWR, catalog number: 631-0133 )
  11. Tin foil
  12. Mice (8-20 days old)
  13. Ethanol (Sigma-Aldrich, catalog number: 32221 )
  14. Acetone (Alfa Aesar, catalog number: L10407 )
  15. Sodium pentobarbitone (Pentoject) (Animalcare, catalog number: XVD-132 )
  16. 5-ethynyl-2’-deoxyuridine (EdU) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: A10044 )
  17. Paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
  18. Phosphate buffered saline (Dulbecco A) (PBS) (Oxoid Limited, catalog number: BR0014 )
  19. Triton X-100 (VWR, catalog number: 28817.295 )
  20. Copper sulphate pentahydrate (CuSO4·5H2O) (VWR, catalog number: 84845.230 )
  21. Biotin-azide (Kerafast, catalog number: EVU101 )
  22. Ascorbic acid (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12950364 )
  23. Streptavidin Alexa555 conjugate (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: S-32355 )
  24. Vectashield mounting medium (Vecta Laboratories, catalog number: H-1000 and H-1200 )
  25. DAPI (4’,6-diamidino-2-phenylindole)
  26. Nail lacquer
  27. Anti-PKD2L-1 (polycystic kidney disease 2-like 1 protein)
  28. PBS tablets
  29. Sodium phosphate monobasic monohydrate (NaH2PO4·H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10667823 )
  30. Sodium phosphate dibasic anhydrous (Na2HPO4) (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10396313 )
  31. Trizma base (Sigma-Aldrich, catalog number: T1503 )
  32. Trizma hydrochloride (Sigma-Aldrich, catalog number: T3253 )
  33. Sucrose (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10386100 )
  34. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: 31437 )
  35. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: 746436 )
  36. Magnesium sulphate (MgSO4·7H2O) (VWR, catalog number: 25165.260 )
  37. Glucose (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10539380 )
  38. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: 21114 )
  39. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, catalog number: D6546 )
  40. Penicillin and streptomycin (Sigma-Aldrich, catalog number: P4458 )
  41. Neurobasal A medium (Thermo Fisher Scientific, GibcoTM, catalog number: 10888-022 )
  42. L-glutamine (Sigma-Aldrich, catalog number: G7513 )
  43. B-27 supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 17504-044 )
  44. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F6178 )
  45. Phosphate buffered saline (PBS) (see Recipes)
  46. 0.2 M phosphate buffer (PB) (see Recipes)
  47. 0.1 M Tris buffer (see Recipes)
  48. Sucrose artificial cerebrospinal fluid (see Recipes)
  49. 8% paraformaldehyde solution (PFA) (see Recipes)
  50. 4% paraformaldehyde solution in 0.1 M PB (see Recipes)
  51. Dissection medium (see Recipes)
  52. Culture medium with serum (see Recipes)
  53. Culture medium serum free (see Recipes)

Equipment

  1. Autoclave (Dixons Surgical Instrument, model: VARIO 1528 )
  2. P1000 pipetman classic pipette (Gilson Scientific, catalog number: F123602 )
  3. P20 pipetman classic pipette (Gilson Scientific, catalog number: F123600 )
  4. Laminar flow hood (biological safety cabinet) (The Baker Company, model: Steril Gard class II type A )
  5. CO2 incubator (Panasonic, model: MCO-18AC-PE )
  6. Dissection microscope (Vickers, instruments)
  7. Dissection equipment (Figure 1)
    1. Dissection scissors
    2. Spring scissors
    3. Fine forceps x 2
    4. Microspatula
  8. Tissue chopper (McIlwain)
  9. Razor blades (Wilkinson sword)
  10. Shaker plate (IKA, model: Vibrax-vxr )
  11. Fine paint brush


    Figure 1. Equipment used for dissection

Procedure

  1. Preparation of organotypic slices
    1. Autoclave dissection equipment, filter paper and tips for Gilson pipettes.
    2. Prepare dissection medium and culture medium in a laminar flow hood.
    3. Pre-acclimatise culture medium to 37 °C in incubator.
    4. Swab the tissue chopper with 70% ethanol and secure a fresh razor blade in place, which has been wiped first with acetone to remove any grease and then swabbed with 70% ethanol.
    5. Make up fresh sucrose artificial cerebrospinal fluid.
    6. Terminally anaesthetise mouse, aged between 8-20 days by an intraperitoneal injection of sodium pentobarbital (60 mg/kg). When all pedal withdrawal reflexes have ceased and there is no response to a strong pinch of the hind limb, nor a corneal reflex, the animal is considered to be sufficiently anaesthetised for the procedure.
    7. Open the chest up and cut away the rib cage to expose the heart. A trans-cardiac perfusion is then performed through the left ventricle with 25 ml of ice cold sucrose artificial cerebrospinal fluid to enhance viability of slices. This is illustrated by Gage et al. (2012).
      Note: This procedure is carried out using fixative and for this protocol, sucrose artificial cerebrospinal fluid is administered via a syringe – a pump system is not required.
    8. Remove the dorsal skin and perform a dorsal laminectomy to expose the spinal cord. Remove the spinal cord using fine spring scissors and fine forceps and place in a 50 mm Petri dish containing ice cold dissection medium (Figure 2). This is procedure is illustrated in a video taken from Gunther et al. (2012).
      Note: In this example the procedure is carried on fixed tissue. For organotypic slice cultures the spinal cord must not be placed in paraformaldehyde solution after it has been dissected free but in cold dissection medium.


      Figure 2. A series of images depicting the dissection of the spinal cord

    9. Remove the meninges (pia, arachnoid and dura mater) under a dissecting microscope, using fine forceps. Select the mid-lower thoracic/upper lumbar region of the spinal cord (approximately T8-L2; Figure 3).


      Figure 3. Removing the meniges from the spinal cord. A. A diagram displaying the meninges (taken from Medical Dictionary, accessed May 2016); B. An image depicting removal of the meninges; C. A diagram displaying the region selected for culture.

    10. Transfer the selected region of spinal cord, using a microspatula, onto a sterile filter paper moistened with dissection medium that is situated on the plate of the tissue chopper and cut 300 µm transverse slices.
    11. Transfer the slices, using a microspatula, to a Petri dish containing ice cold dissection medium, this will remove any excitotoxic chemicals and debris.
    12. Under a dissection microscope using fine forceps and a microspatula, separate the slices and discard any damaged slices.
      Note: All procedures from this point onwards are performed in a laminar flow hood.
    13. Pipette 1 ml of pre-acclimatised culture medium into each well of a sterile 6-well culture plate and, using sterile forceps, place a millicell organotypic filter insert on top of the culture medium.
    14. Immediately following this, using a Gilson P1000 pipette with a sterile tip that has had the end cut off to widen the diameter (thus limiting damage to the slices), transfer 2 to 6 slices onto each filter. Care must be taken that the slices are not touching each other or the side of the filter. Remove all excess medium from around the slice by means of a Gilson pipette with sterile tip; ensure there is no medium above the filter (Figure 4).


      Figure 4. Method for preparing organotypic slice culture

    15. Transfer culture plate to the incubator, where slices are maintained at 37 °C under standard conditions; 100% humidity, 95% air, 5% CO2 enriched environment.
    16. After 24 h in culture, using sterile forceps to lift the organotypic filter insert carefully out of the well, remove culture medium using Gilson P1000 pipette with a sterile tip and replace the culture medium with 1 ml of serum-free medium using Gilson P1000 pipette with a sterile tip. Carefully place filter back onto the fresh medium, ensuring that there are no air bubbles under the filter. The serum is required initially, as it aids the slices in recovery from the trauma of explantation. The key advantage of changing to serum free medium is that the exact constituents can be determined. Serum may also contain substances that influence the growth of a particular population of cells and may promote microglial proliferation (Coltman and Ide, 1996).
    17. For the remaining time in culture, remove half the medium (0.5 ml) and replace with 0.5 ml of fresh serum free medium, every second day. This technique leaves some of the natural growth factors, which assists in maintaining good viability in the cultured slices. The cultured slices maintain good morphology (Figure 5).


      Figure 5. An organotypic spinal cord slice growing in culture

  2. Addition of 5-ethynyl-2’-deoxyuridine (EdU) to organotypic cultures
    1. After 48 h in culture, add 1 µl of EdU (1 mM) directly to the 1 ml of medium in each well. Add fresh EdU (0.5 µl) following any subsequent medium changes. EdU is a thymidine analogue that is incorporated into DNA during active synthesis in the S-phase of the cell cycle (Figure 6) (Chehrehasa et al., 2009) providing a fast and reproducible method to employ for the study of cell proliferation (Figure 7).
      Note: This stage can be omitted if you do not wish to investigate cell proliferation.


      Figure 6. EdU is incorporated into the newly synthesised DNA and visualised with addition of click chemistry and a fluorescent azide


      Figure 7. Timeline for culturing slices.
      Schematic of the timing for culturing slices and addition of EdU.

    2. After the allotted time in culture, fix the slices by removing the filter from the 6-well culture plate, with slices in situ and place on top of 1 ml of 4% paraformaldehyde in 0.1 M phosphate buffer in a new 6-well culture plate. Carefully apply a further 500 µl of the 4% paraformaldehyde solution on top of the filter, covering the slices completely.
    3. Leave slices in fixative for 6 h at 4 °C.
    4. Using a fine paint brush, carefully ease the slice free from the filter and place individually in 500 µl 0.1 M phosphate buffered saline in a 24-well histology tray.
      Note: Slices can be stored at 4 °C in phosphate buffered saline for up to 7 days at this point.

  3. EdU detection in organotypic cultures
    Slices are kept in the 24-well histology tray throughout and this technique of free floating the slices is continued during the procedure. Each solution is removed from the well carefully at the end of allotted time using a pipette and the next solution carefully added by allowing it to run down the side of the well and not directly on top of the delicate slices. Unless otherwise stated, all steps are carried out at room temperature, with gentle agitation of the sections on a shaker plate.
    1. Incubate in 500 µl PBS with 0.2 % triton for 12 min.
    2. Wash 2 x 10 min in 500 µl 0.1 M Tris buffer.
    3. Incubate slices in a mix of 320 µl distilled water, 25 µl Tris buffer (2 M), 50 µl copper sulphate (10 mM), 5 µl biotinylated azide (1 mM) and 100 µl ascorbic acid (0.5 M) in each well (make up fresh each time) for 30 min.
    4. Wash 2 x 10 min in 500 µl 0.1 M Tris buffer.
    5. Wash 1 x 10 min in 500 µl PBS.
    6. Incubate in Streptavidin Alexa555 1:1,000 in 500 µl PBS with 0.1 % triton for 1 h, protect from light by wrapping histology tray in tin foil for this and any subsequent steps.
    7. Wash 3 x 10 min in 500 µl PBS.
    8. The sections can then be immunohistochemically labelled with appropriate antibodies following instructions of the supplier at this stage before mounting (Figure 8).
      Note: The slices are not agitated for longer than 2 h in the primary antibodies, as they start to disintegrate. If longer incubation time is required leave in the histology tray without agitation at 4 °C.
    9. Free float the slices in a Petri dish of distilled water and carefully float the slice onto a glass slide and guide into position with a fine paint brush.
    10. Protect from light and allow to air dry.
    11. Mount using VectaShield mounting media with or without DAPI (4’,6-diamidino-2-phenylindole).
    12. Apply glass coverslip and seal with nail lacquer.

Representative data

Following detection of EdU, these slices were also immunohistochemically labelled with anti-PKD2L-1 (polycystic kidney disease 2-like 1 protein) labelling cerebrospinal contacting neurones.


Figure 8. An example taken of EdU labelling in the region surrounding the central canal of an organotypic cultured spinal cord slice 

Notes

Minimising the time taken for the spinal cord to be removed from the animal and established in culture is imperative to achieving good viability in the slices. Therefore the preparation must be carried out as efficiently as possible, while still ensuring there is no damage to the spinal cord.

Recipes

  1. Phosphate buffered saline (PBS)
    Dissolve 10 PBS tablets in 1 L distilled water (pH 7.4).
  2. 0.2 M phosphate buffer (PB)
    Dissolve 9.35 g NaH2PO4·2H2O in 300 ml distilled water.
    Dissolve 33.85 g Na2HPO4 in 1,200 ml distilled water.
    Add NaH2PO4·2H2O solution to Na2HPO4 solution until pH is 7.4.
  3. 0.1 M Tris buffer
    0.695 g Trizma base
    3.03 g Trizma hydrochloride
    500 ml distilled water
    Note: Adjust to pH 7.6 with a few drops of sodium hydroxide if necessary.
  4. Sucrose artificial cerebrospinal fluid
    74.5 g sucrose
    2.184 g NaHCO3
    0.2237 g KCl
    0.4933 g MgSO4·7H2O
    0.39 g NaH2PO4
    1.8 g glucose
    1 ml of 1 M CaCl2 solution
    1 L distilled water
    Note: Add all ingredients except CaCl2 to the distilled water, shake to dissolve completely then add CaCl2 at the end and shake to disperse.
  5. 8% paraformaldehyde solution (PFA)
    80 g paraformaldehyde
    1 L of distilled water
    Note: Gentle heat and continual stirring may be needed to help dissolve the paraformaldehyde; this must be carried out in a fume hood. Solution becomes cloudy, so clear with a few drops of 1 M sodium hydroxide solution, if necessary adjust the pH to 7.2 with a small amount of dilute HCl.
  6. 4% paraformaldehyde solution in 0.1 M PB
    50 ml 8% PFA
    50 ml 0.2 M PB
  7. Dissection medium
    Dulbecco’s modified Eagles medium
    100 fold dilution of penicillin and streptomycin (Stock solution: 5,000 units’ penicillin, 5 mg streptomycin)
  8. Culture medium with serum
    Neurobasal A medium
    100 fold dilution L-glutamine (Stock solution: 200 mM)
    100 fold dilution of penicillin and streptomycin (Stock solution: 5,000 units’ penicillin, 5 mg streptomycin)
    10 fold dilution of fetal bovine serum
  9. Culture medium serum free
    Neurobasal A medium
    50 fold dilution B27 supplement
    100 fold dilution L-glutamine (Stock solution: 200 mM)
    100 fold dilution of penicillin and streptomycin (Stock solution: 5,000 units’ penicillin, 5 mg streptomycin)

Acknowledgments

This procedure known as the interface method was modified for spinal cord culture from the original method of Stoppini et al. (1991).

References

  1. Chehrehasa, F., Meedeniya, A. C., Dwyer, P., Abrahamsen, G. and Mackay-Sim, A. (2009). EdU, a new thymidine analogue for labelling proliferating cells in the nervous system. J Neurosci Methods 177(1): 122-130.
  2. Coltman, B. W. and Ide, C. F. (1996). Temporal characterization of microglia, IL-1β-like immunoreactivity and astrocytes in the dentate gyrus of hippocampal organotypic slice cultures. Int J Dev Neurosci 14(6): 707-719.
  3. Gage, G. J., Kipke, D. R. and Shain, W. (2012). Whole animal perfusion fixation for rodents. J Vis Exp(65).
  4. Gunther, R., Suhr, M., Koch, J. C., Bahr, M., Lingor, P. and Tonges, L. (2012). Clinical testing and spinal cord removal in a mouse model for amyotrophic lateral sclerosis (ALS). J Vis Exp(61).
  5. Stoppini, L., Buchs, P. A. and Muller, D. (1991). A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37(2): 173-182.

材料和试剂

  1. 滤纸,55mm(Sigma-Aldrich,Whatman ,目录号:WHA1441055)
  2. Millicell器官型滤器插件0.4μm,30mm(EDM Millipore,目录号:Picmorg50)
  3. 6孔培养皿(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:140675)
  4. 24孔组织学托盘(Sigma-Aldrich,CELLSTAR ,目录号:662160)
  5. 50mm灭菌培养皿(Camlab,目录号:p17118) 注意:此链接会导致一系列培养皿。
  6. 移液管吸头,1,000μl(SARSTEDT,目录号:70.762.010)
  7. 移液管吸头,200μl(SARSTEDT,目录号:70.760.012)
  8. 注射器
  9. 玻璃显微镜载玻片(Academy Science,目录号:N/A143)
  10. 玻璃盖玻片(VWR,目录号:631-0133)
  11. 锡箔
  12. 小鼠(8-20天龄)
  13. 乙醇(Sigma-Aldrich,目录号:32221)
  14. 丙酮(Alfa Aesar,目录号:L10407)
  15. 戊巴比妥钠(Pentoject)(Animalcare,目录号:XVD-132)
  16. (Edu)(Thermo Fisher Scientific,Molecular Probes ,目录号:A10044)的5'-乙炔基-2'-脱氧尿苷
  17. 多聚甲醛(Sigma-Aldrich,目录号:158127)
  18. 磷酸盐缓冲盐水(Dulbecco A)(PBS)(Oxoid Limited,目录号:BR0014)
  19. Triton X-100(VWR,目录号:28817.295)
  20. 硫酸铜五水合物(CuSO 4·5H 2 O)(VWR,目录号:84845.230)
  21. 生物素 - 叠氮化物(Kerafast,目录号:EVU101)
  22. 抗坏血酸(Thermo Fisher Scientific,Fisher Scientific,目录号:12950364)
  23. 链霉亲和素Alexa 555 缀合物(Thermo Fisher Scientific,Molecular Probes ,目录号:S-32355)
  24. Vectashield封固剂(Vecta Laboratories,目录号:H-1000和H-1200)
  25. DAPI(4',6-二脒基-2-苯基吲哚)
  26. 指甲油
  27. 抗PKD2L-1(多囊肾病2样1蛋白)
  28. PBS平板电脑
  29. 磷酸二氢钠一水合物(NaH 2 PO 4 H 2·H 2 O)(Thermo Fisher Scientific,Fisher Scientific,目录号:10667823)
  30. 磷酸氢二钠无水(Na 2 HPO 4)(Thermo Fisher Scientific,Fisher Scientific,目录号:10396313)
  31. Trizma碱(Sigma-Aldrich,目录号:T1503)
  32. 盐酸三甲胺(Sigma-Aldrich,目录号:T3253)
  33. 蔗糖(Thermo Fisher Scientific,Fisher Scientific,目录号:10386100)
  34. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:31437)
  35. 氯化钾(KCl)(Sigma-Aldrich,目录号:746436)
  36. 硫酸镁(MgSO 4·7H 2 O)(VWR,目录号:25165.260)
  37. 葡萄糖(Thermo Fisher Scientific,Fisher Scientific,目录号:10539380)
  38. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:21114)
  39. Dulbecco改良的Eagle培养基(DMEM)(Sigma-Aldrich,目录号:D6546)
  40. 青霉素和链霉素(Sigma-Aldrich,目录号:P4458)
  41. Neurobasal A培养基(Thermo Fisher Scientific,Gibco TM ,目录号:10888-022)
  42. L-谷氨酰胺(Sigma-Aldrich,目录号:G7513)
  43. B-27补充物(Thermo Fisher Scientific,Gibco TM ,目录号:17504-044)
  44. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F6178)
  45. 磷酸盐缓冲盐水(PBS)(见Recipes)
  46. 0.2 M磷酸盐缓冲液(PB)(参见配方)
  47. 0.1 M Tris缓冲液(见配方)
  48. 蔗糖人工脑脊液(见配方)
  49. 8%多聚甲醛溶液(PFA)(参见配方)
  50. 4%多聚甲醛在0.1M PB中的溶液(参见配方)
  51. 解剖介质(见配方)
  52. 含血清培养基(见配方)
  53. 培养基无血清(见配方)

设备

  1. 高压灭菌器(Dixons Surgical Instrument,型号:VARIO 1528)
  2. P1000移液管经典移液管(Gilson Scientific,目录号:F123602)
  3. P20移液管经典移液管(Gilson Scientific,目录号:F123600)
  4. 层流罩(生物安全柜)(贝克公司,型号:Steril Gard II类A型)
  5. CO 2培养箱(Panasonic,型号:MCO-18AC-PE)
  6. 解剖显微镜(Vickers,instrument)
  7. 解剖设备(图1)
    1. 解剖剪刀
    2. 弹簧剪刀
    3. 精细镊子x 2
    4. 微风
  8. 组织切碎机(McIlwain)
  9. 剃刀刀片(Wilkinson剑)
  10. 振动板(IKA,型号:Vibrax-vxr)
  11. 精彩画笔


    图1.用于解剖的设备

程序

  1. 器官切片的制备
    1. 高压灭菌夹层设备,滤纸和Gilson移液器的提示
    2. 在层流罩中准备解剖介质和培养基
    3. 将预适应培养基在培养箱中37℃培养
    4. 用70%乙醇擦拭组织切碎器,并固定新鲜的剃刀刀片到位,首先用丙酮擦拭,以去除任何油脂,然后用70%乙醇擦拭。
    5. 补充新鲜蔗糖人工脑脊液
    6. 终末麻醉小鼠,通过腹膜内注射戊巴比妥钠(60mg/kg)在8-20天之间老化。当所有的脚踝退缩反射已经停止并且对后肢的强烈的掐捏没有反应,也没有角膜反射时,认为该动物对于该过程被充分麻醉。
    7. 打开胸部,切除肋骨,露出心脏。然后通过左心室用25ml冰冷的蔗糖人工脑脊液进行经心脏灌注,以增强切片的活力。这由Gage等人说明。 (2012)。
      注意:此程序使用固定剂进行,对于该方案,蔗糖人工脑脊液通过注射器给药 - 不需要泵系统。
    8. 去除背部皮肤,进行背椎间盘切除术暴露脊髓。删除脊髓使用细弹簧剪刀和细镊子,并放置在含有冰冷的解剖介质的50毫米培养皿(图2)。这个过程在取自Gunther等人的视频中示出。 (2012)。
      注意:在本例中,程序在固定组织上进行。对于器官切片培养物,脊髓在解剖自由但在冷剥离介质中后,不能放置在多聚甲醛溶液中。


      图2.描绘脊髓切开的一系列图像

    9. 在解剖显微镜下,使用细镊子除去脑膜(脑,蛛网膜和硬脑膜)。选择脊髓的中下胸部/上腰部(大约T8-L2;图3)。


      图3.从脊髓中取出小叶。 A.显示脑膜的图表(摘自2016年5月访问的"医学词典"); B.描绘去除脑膜的图像; C.显示选择用于培养的区域的图。

    10. 使用微滴管将选定的脊髓区域转移到用位于组织切碎器的板上的切割介质润湿的无菌滤纸上,并切割300μm的横切片。
    11. 转移切片,使用小刮刀,到含有冰冷的解剖介质的培养皿,这将删除任何兴奋性毒性化学品和碎片。
    12. 在使用细镊子和小刮刀的解剖显微镜下,分离切片并丢弃任何损伤的切片 注意:从这一点开始的所有程序都在层流罩中进行。
    13. 吸取1毫升预驯化培养基到无菌6孔培养板的每个孔,并使用无菌钳,在培养基的顶部放置millicell器官型滤器插入。
    14. 紧接着,使用具有无菌末端的Gilson P1000移液管,其末端被切除以加宽直径(从而限制对切片的损伤),将2至6个切片转移到每个过滤器上。必须注意,切片不彼此接触或过滤器的侧面。用无菌尖端的Gilson移液管从切片周围除去所有多余的培养基;确保过滤器上方没有介质(图4)。


      图4.制备器官切片培养的方法

    15. 转移培养板到孵化器,其中切片在标准条件下保持在37°C; 100%湿度,95%空气,5%CO 2富集环境
    16. 培养24小时后,使用无菌镊子将器官型滤器小心地从孔中取出,使用Gilson P1000移液管用无菌尖取出培养基,并使用Gi??lson P1000移液管用1ml无血清培养基替换培养基无菌尖端。小心地将过滤器放回新鲜介质,确保过滤器下没有气泡。最初需要血清,因为它帮助切片从外植创伤中恢复。改变为无血清培养基的关键优点是可以确定确切的成分。血清还可以包含影响特定细胞群生长的物质,并且可以促进小胶质细胞增殖(Coltman和Ide,1996)。
    17. 对于培养中的剩余时间,取出一半培养基(0.5ml),并每隔一天更换0.5ml新鲜无血清培养基。这种技术留下一些天然生长因子,这有助于在培养的切片中维持良好的活力。培养的切片保持良好的形态(图5)。


      图5.在培养物中生长的器官性脊髓切片

  2. 向器官型培养物中加入5-乙炔基-2'-脱氧尿苷(EdU)
    1. 培养48小时后,加入1μl的EdU(1mM)直接到每个孔的1ml培养基中。在任何后续培养基更换后加入新鲜EdU(0.5μl)。 EdU是胸苷类似物,其在细胞周期的S期中的活性合成期间并入DNA中(图6)(Chehrehasa等人,2009),提供了快速和可重复的方法,用于研究细胞增殖(图7) 注意:如果您不想调查细胞增殖,可以省略此阶段。


      图6.将EdU并入新合成的DNA中,并添加点击化学和荧光叠氮化物进行可视化


      图7.培养切片的时间线。 培养切片和添加EdU的时间示意图。

    2. 在培养物中分配的时间后,通过从6孔培养板中原位取出滤膜来固定切片并置于1ml的4%多聚甲醛的0.1M磷酸盐缓冲液一个新的6孔培养板。小心地在过滤器的顶部应用另外500μl的4%多聚甲醛溶液,完全覆盖切片
    3. 在4℃下将切片在固定液中保持6小时
    4. 使用精细的油漆刷,小心地将切片从过滤器中取出,并单独放置在24孔组织学托盘中的500μl0.1 M磷酸盐缓冲盐水中。
      注意:切片可以在4°C下储存在磷酸盐缓冲液中,最多7天。

  3. 器官型培养中的EdU检测
    切片保持在24孔组织学托盘中,并且在手术期间继续这种自由浮动切片的技术。在分配时间结束时使用移液管小心地从孔中移出每种溶液,并且通过允许其沿着孔的侧面跑下而不直接在精细切片的顶部上而小心地添加下一个溶液。除非另有说明,所有步骤在室温下进行,在振动板上轻轻搅拌各部分
    1. 在含有0.2%triton的500μlPBS中孵育12分钟
    2. 在500μl0.1M Tris缓冲液中洗涤2×10分钟。
    3. 在每个孔中混合320μl蒸馏水,25μlTris缓冲液(2M),50μl硫酸铜(10mM),5μl生物素化叠氮化物(1mM)和100μl抗坏血酸(0.5M)的混合物中孵育切片每次补充新鲜)30分钟。
    4. 在500μl0.1M Tris缓冲液中洗涤2×10分钟
    5. 在500μlPBS中洗涤1×10分钟
    6. 在500μl含有0.1%triton的PBS中的链霉抗生物素蛋白Alexa 555 1:1,000中孵育1小时,通过将组织学托盘包裹在锡箔中以避免光照,以及随后的步骤。
    7. 在500μlPBS中洗涤3×10分钟。
    8. 然后可以根据供应商在安装之前的这个阶段的说明用合适的抗体对切片进行免疫组织化学标记(图8)。
      注意:在第一抗体中,切片不搅拌超过2小时,因为它们开始崩解。如果需要更长的孵育时间,在组织学托盘中在4℃不搅拌。
    9. 自由漂浮在培养皿中的蒸馏水的切片,并小心地将切片漂浮在玻璃载片上,并用一个精细的油漆刷引导到位。
    10. 避光保存,让空气干燥。
    11. 使用带或不带DAPI(4',6-二脒基-2-苯基吲哚)的VectaShield封固介质进行安装。
    12. 应用玻璃盖片和用指甲油密封。

代表数据

在检测EdU后,这些切片也用标记脑脊髓接触神经元的抗PKD2L-1(多囊肾病2样1蛋白)免疫组织化学标记。


图8.在围绕器官型培养的脊髓切片的中央管的区域中的EdU标记的实例</strong>

笔记

最小化脊髓从动物中移除并在培养物中建立所花费的时间对于在切片中获得良好的活力是必需的。因此,必须尽可能有效地进行制备,同时仍然确保脊髓没有损伤。

食谱

  1. 磷酸盐缓冲盐水(PBS)
    将10个PBS片剂溶解在1L蒸馏水(pH7.4)中
  2. 0.2 M磷酸盐缓冲液(PB)
    将9.35g NaH 2 PO 4·2H 2·2H 2溶于300ml蒸馏水中。
    在1,200ml蒸馏水中溶解33.85g Na 2 HPO 4。
    将NaH 2 PO 4 4·2H 2 O溶液加入到Na 2 HPO 4水溶液中,溶液,直至pH为7.4。
  3. 0.1 M Tris缓冲液
    0.695克Trizma碱
    3.03g盐酸三甲胺 500毫升蒸馏水
    注意:如有必要,用几滴氢氧化钠调节至pH 7.6。
  4. 蔗糖人工脑脊液
    74.5克蔗糖 2.184g NaHCO 3·
    0.2237克KCl
    0.4933g MgSO 4 7H 2 O 2·h/v 0.39g NaH 2 PO 4 sub/
    1升蒸馏水
    注意:将除了CaCl 2 2以外的所有成分添加到蒸馏水中,摇动以完全溶解,然后在末端加入CaCl 2并摇动分散。
  5. 8%多聚甲醛溶液(PFA) 80g多聚甲醛
    1升蒸馏水
    注意:可能需要温和加热和连续搅拌以帮助溶解多聚甲醛;这必须在通风橱中进行。溶液变浑浊,用几滴1M氢氧化钠溶液澄清,如有必要,用少量稀HCl调节pH至7.2。
  6. 4%多聚甲醛在0.1M PB中的溶液 50 ml 8%PFA
    50ml 0.2M PB
  7. 解剖介质
    Dulbecco's modified Eagles培养基
    青霉素和链霉素的100倍稀释液(母液:5,000单位青霉素,5mg链霉素)
  8. 含血清培养基
    Neurobasal A介质
    100倍稀释的L-谷氨酰胺(储备溶液:200mM) 青霉素和链霉素的100倍稀释液(母液:5,000单位青霉素,5mg链霉素)
    胎牛血清稀释10倍
  9. 培养基无血清
    Neurobasal A介质
    稀释50倍B27补充剂
    100倍稀释的L-谷氨酰胺(储备溶液:200mM) 青霉素和链霉素的100倍稀释液(母液:5,000单位青霉素,5mg链霉素)

致谢

这种被称为界面方法的方法被修改为用于脊髓培养从Stoppini等人的原始方法。 (1991)。

参考文献

  1. Chehrehasa,F.,Meedeniya,AC,Dwyer,P.,Abrahamsen,G.和Mackay-Sim,A。(2009)。  EdU,一种用于在神经系统中标记增殖细胞的新的胸腺嘧啶类似物。 J Neurosci Methods :122-130。
  2. Coltman,BW和Ide,CF(1996)。  Temporal表征海马器官型切片培养物的齿状回中的小胶质细胞,IL-1β样免疫反应性和星形胶质细胞。 Int J Dev Neurosci 14(6):707-719。
  3. Gage,GJ,Kipke,DR和Shain,W。(2012)。  对于啮齿动物的全动物灌注固定。 (65)。
  4. Gunther,R.,Suhr,M.,Koch,JC,Bahr,M.,Lingor,P。和Tonges,L.(2012)。  用于肌萎缩性侧索硬化(ALS)的小鼠模型中的临床测试和脊髓切除术 em>(61)。
  5. Stoppini,L.,Buchs,PA和Muller,D。(1991)。  用于神经组织的器官培养的简单方法。 J Neurosci Methods 37(2):173-182。
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How to cite this protocol: Daniel, J. M., Deuchars, J. and Deuchars, S. A. (2016). Organotypic Spinal Cord Slice Cultures and a Method to Detect Cell Proliferation in These Slices . Bio-protocol 6(19): e1951. DOI: 10.21769/BioProtoc.1951; Full Text



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