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Aug 2020
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Enrichment of Vascular Fragments from Mouse Embryonic Brains for Endothelial Cell Analysis
用于内皮细胞分析小鼠胚胎脑血管片段富集   

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Abstract

Endothelial cells in the brain interact with other cell types, forming the blood-brain barrier. This barrier controls the movement of solutes into and out of the brain, regulating pathophysiological processes and drug delivery to the brain. Common isolation methods used to study these cells during embryonic development involve enzymatic treatment and cell sorting using specific markers. This process modifies the cell state and produces minute amounts of sample. Here, we describe a protocol for the enrichment of vascular cells from embryonic brains based on dextran separation. In this method, the brain is lightly disrupted with a pestle and then resuspended in a dextran solution. Low-speed centrifugation permits the separation of the parenchymal and vascular fractions. Further centrifugation steps improve fractionation. This method is simple and fast and produces enough sample for biochemical assays.


Graphic abstract:



Purification of vascular fragments from an embryonic brain


Keywords: Embryo (胚胎), Brain (大脑), Vasculature (脉管系统), Dextran (葡萄聚糖), Cholesterol (胆固醇)

Background

Endothelial cells in the brain form a specialized barrier that limits and controls the passage of large and small molecules from the bloodstream into the brain (Zlokovic, 2008). In their natural niche, brain endothelial cells interact closely with several cell types, including pericytes, glia, and immune cells, which are integral to brain homeostasis (Armulik et al., 2005; Gavins et al., 2007). The study of brain endothelial cell biology has benefited greatly from the use of procedures to isolate either endothelium or vascular complexes, which include endothelial cells, pericytes, and glial contacts (radial glia, oligodendrocytes, and astrocyte endfeet). The former is normally achieved by fluorescence-activated cell sorting (FACS) isolation (Daneman et al., 2010), whereas the latter is done by centrifugation in a dextran solution (Yousif et al., 2007). FACS-based isolation can alter the properties of endothelial cells and is expensive and inefficient, leading to the isolation of very few cells that may not reflect their native state. These issues are more evident when obtaining samples from embryonic brains, where the sample is extremely limited, and fewer than 5000 endothelial cells are typically obtained from one brain. This number is adequate for transcriptional analyses using specialized kits (Santander et al., 2020) but is insufficient for biochemical assays. Conversely, dextran-based isolation of vascular complexes has not been applied to embryonic brains because the standard protocol requires a much bigger sample obtained from single mouse embryos. The ability to obtain enough sample for biochemical assays from single embryonic brains is necessary for the study of developmental vascular diseases, such as the Fowler syndrome (Santander et al., 2020). Here, to overcome these sample size issues, we describe the adaptation of the paradigm of dextran-based isolation of vascular fragments to individual mouse embryo brains. We demonstrate the utility of vascular fragment isolation to measure cholesterol content in embryonic brain blood vessels, an assay not feasible using FACS-isolated endothelial cells.

Materials and Reagents

  1. 15 ml conic bottom tube

  2. DuraSeal laboratory film (Sigma, catalog number: D3172)

  3. Polypropylene 1.5 ml tubes

  4. Assay plate, 96-well, black

  5. Assay plate, 96-well, clear

  6. PVDF membrane (Thermo, catalog number: 88520)

  7. Culture plate

  8. Female and male mice

  9. PBS 10× (Apex, catalog number: 20-134)

  10. Dextran (70 kDa Highly hygroscopic, should be maintained in an inert atmosphere) (TCI, catalog number: D1449)

  11. IGEPAL (Sigma, catalog number: I8896)

  12. Sodium dodecyl sulfate (Sigma, catalog number: L3771)

  13. Protease inhibitor cocktail (Thermo, catalog number: 78429)

  14. MilliQ water

  15. BCA Protein Assay kit (Thermo, catalog number: 23225)

  16. Methanol (Merck, catalog number: 1060091000)

  17. Chloroform (Merck, catalog number: 1070242500)

  18. Amplex Red Cholesterol Assay Kit (Thermo, catalog number: A12216)

  19. Protein loading buffer 4× (Bio-Rad, catalog number: 1610747)

  20. 2-mercaptoethanol (Sigma, catalog number: M6250)

  21. Buffer Tris-Glycine 10× (Apex, catalog number: 18-238B)

  22. Pre-cast 10% polyacrylamide gel (Bio-Rad, catalog number: 4561033)

  23. Powdered skim milk

  24. TBST 10× (Apex, catalog number: 18-235B)

  25. Rabbit anti-ERG (Abcam, catalog number: ab92513)

  26. Rabbit anti-PECAM1 (Santa Cruz, catalog number: sc-1506)

  27. Rabbit anti-PAX6 (Millipore, catalog number: AB2237)

  28. Rabbit anti-TBR1 (Abcam, catalog number: ab31940)

  29. Mouse anti-ACTINB (R&D Systems, catalog number: MAB8929)

  30. Donkey anti-rabbit IGG (Thermo, catalog number: A16029)

  31. Donkey anti-mouse IGG (Thermo, catalog number: A16011)

  32. Chemiluminescence kit (Bio-Rad, catalog number: 1705061)

  33. PBS 1× (see Recipes)

  34. 18% Dextran (see Recipes)

  35. Lysis buffer (see Recipes)

  36. Running buffer (see Recipes)

  37. Transfer buffer (see Recipes)

  38. TBST 1× (see Recipes)

Equipment

  1. Dumont #5 fine forceps (Fine Science Tools, catalog number: 11251-30)

  2. Tube pestle (Sigma, catalog number: Z359947)

  3. CO2 euthanasia chamber

  4. Dissection microscope

  5. Refrigerated microcentrifuge

  6. Heating block

  7. Fume hood

  8. Gel documentation system

Software

  1. LibreOffice Calc

  2. R

Procedure

  1. Embryo brain dissection

    1. Breed female and male mice in a 2:1 or 1:1 ratio.

    2. Check females for the presence of a vaginal plug every day within the first hour of the light cycle.

    3. Separate females with a plug to avoid re-plugging and register that morning as embryonic day (E) 0.5.

    4. On day E15.5, euthanize females following institutional guidelines. We use a combination of CO2 asphyxiation and cervical dislocation. See Note 1.

    5. Remove the uteri, place them in PBS 1×, and separate each implantation site. Care should be taken to maintain the yolk sac intact to minimize tissue damage.

    6. Under a dissection microscope, dissect the embryo free of extraembryonic tissues. If genotyping is required, cut the tip of the tail or a paw and place it in a tube. See Note 2.

    7. Cut the embryo head and peel the skin and meningeal membranes covering the brain using Dumont #5 fine forceps. See Note 3 and Figure 1.



      Figure 1. Dissection of a mouse embryo brain. The head skin is peeled away along the dotted line using Dumont #5 forceps (Left). The meningeal layers covering the brain are removed carefully to expose the brain (Center, dotted line). Pushing the brain gently from the side releases the whole organ from the head (Right).


    8. Remove the brain from the head by cutting the rhombencephalon at the base of the skull and gently pushing the forebrain from the side.

    9. Place the brain in 1 ml of ice-cold PBS in a 1.5 ml polypropylene tube.


  2. Vascular fragment isolation (all steps should be performed on ice)

    1. Disrupt brains with five strokes using a sterile 1.5 ml tube pestle.

    2. Centrifuge at 1,500 × g for 20 min at 4°C.

    3. During centrifugation, prepare enough 18% dextran solution in PBS to individually process each of the brains collected.

    4. Discard the supernatant and resuspend the pellet in 500 μl of 18% dextran. See Note 4.

    5. Centrifuge at 1,500 × g for 20 min at 4°C.

    6. Transfer the supernatant to another tube. See Note 5.

    7. Resuspend the pellet in 500 μl of 18% dextran.

    8. Centrifuge both fractions at 1,500 × g for 20 min at 4°C. Pellets are the vascular fragments.

    9. Transfer the supernatant to a different tube and add 1 ml of PBS.

    10. Centrifuge the diluted supernatants at 10,000 × g for 10 min at 4°C and discard the supernatant. The pellets are the parenchymal fractions.

    11. Store vascular and parenchymal fractions at < -70°C until use.


  3. Western blotting for the evaluation of enrichment of fractions

    1. Add 100 μl of lysis buffer directly to the pellet on ice. Care should be taken to maintain the pellet frozen until the lysis buffer is added by keeping the tubes on dry ice or liquid nitrogen.

    2. Lyse the sample by pipetting up and down until the lysate looks homogeneous. See Note 6.

    3. Pool pellets corresponding to the same fractions of the same sample by transferring all the lysate from the first tube to the second. Lyse the second pellet by pipetting. See Note 7.

    4. Transfer 10 μl of lysate to a different 1.5 ml tube for protein quantification. Keep at < -70°C.

    5. Repeat Steps C1-C4 for each sample to be analyzed in parallel. Always process controls alongside experimental samples.

    6. Add 2-mercaptoethanol to loading buffer to a concentration of 5%.

    7. Mix an aliquot of 15 μg of protein with 1/3 volume of loading buffer with 2-mercaptoethanol and heat at 98°C for 5 min.

    8. Load into a 10% polyacrylamide gel and run for the appropriate time to resolve proteins in the range of 30 kDa and 150 kDa in Running buffer.

    9. Incubate a PVDF membrane in methanol for 5 min for activation.

    10. Transfer proteins into the activated PVDF membrane at 300 mA for 1 h in Transfer buffer.

    11. Block the membrane by incubating 2 h in 5% skim milk, 0.01% Tween 20 in TBS 1×.

    12. Incubate the membrane with the primary antibody diluted 1:2,000 in blocking buffer.

    13. Wash the membrane three times for 10 min in 0.01% Tween 20 in TBS 1×.

    14. Incubate with the secondary antibody diluted 1:10,000 in blocking buffer.

    15. Wash the membrane three times for 10 min in 0.01% Tween 20 in TBS 1×.

    16. Mix the solutions from the chemiluminescence kit and cover the membrane for 1 min.

    17. Blot excess solution and image in a gel documentation system.

    18. A representative blot demonstrating the enrichment of endothelial markers in the vascular fraction and of a neuronal progenitor marker in the parenchymal fraction is shown in Figure 2.



      Figure 2. Enrichment of vascular and parenchymal fractions. Western blot analysis of endothelial (PECAM1, ERG) and neuronal progenitor (PAX6, TBR1) markers in the vascular (V) and parenchymal (P) fractions. Note that the PAX6 signal is detected in vascular fragments, representing PAX6 expression in brain endothelial cells (Vasudevan et al., 2008) and/or vascular adherent neuro/glial progenitors (Tan et al., 2016).


  4. Lipid extraction

    1. Add 100 μl of lysis buffer directly to the pellet on ice. Care should be taken to maintain the pellet frozen until lysis buffer is added by keeping the tubes on dry ice or liquid nitrogen.

    2. Lyse the sample by pipetting up and down until the lysate looks homogeneous. See Note 6.

    3. Pool pellets corresponding to the same fractions of the same sample by transferring all the lysate from the first tube to the second. Lyse the second pellet by pipetting. See Note 7.

    4. Transfer 10 μl of lysate to a different 1.5 ml tube for protein quantification. Keep at < -70°C.

    5. Repeat Steps D1-D4 for each sample to be analyzed in parallel. Always process controls alongside experimental samples.

    6. In a fume hood, add 400 μl of methanol to the 90 μl of lysate, followed by 800 μl of chloroform. See Note 8.

    7. Seal tubes with DuraSeal laboratory film. See Note 9.

    8. Vortex each tube for 1 min to mix both phases well.

    9. Heat at 60°C for 30 min using a heating block or bath.

    10. Vortex for 1 min.

    11. Incubate overnight at 4°C to allow phases to separate.

    12. Centrifuge at 1,500 × g for 20 min at 4°C.

    13. Transfer 400 μl of the hydrophobic (lower) phase into a new tube. To avoid contaminating the organic phase with the aqueous one, push some air into the pipette tip before introducing it into the solution, and release it in the upper part of the lower phase.

    14. Transfer 50 μl of organic phase into a new tube and evaporate under an inert atmosphere. After evaporation, no crystallization should be visible. Two tubes should be prepared in this manner for each sample to measure cholesterol in duplicate.


  5. Cholesterol measurement

    1. Prepare all reagents in the Amplex Red Cholesterol Assay Kit, following the manufacturer’s instructions.

    2. Add 50 μl of Reaction buffer 1× to the tubes containing the evaporated organic phase.

    3. Vortex for 1 min. See Note 10.

    4. Transfer to a black 96-well plate.

    5. Construct a cholesterol standard curve following the manufacturer’s instructions. Transfer 50 μl to the black 96-well plate.

    6. Set up the reaction mixture as described by the manufacturer and add 50 μl to each well containing the samples and the curve.

    7. Incubate at 37°C for 30 min.

    8. Measure the fluorescence with excitation at 560 nm and emission at 590 nm.


  6. Protein measurement

    1. Thaw sample aliquots on ice and prepare 100 μl of a 10-fold dilution by adding 90 μl of MilliQ water to each tube.

    2. Construct a BSA curve by diluting the BSA standard as instructed by the manufacturer.

    3. Transfer 25 μl of each sample and curve point to a clear 96-well plate.

    4. Prepare enough reaction mixture for all samples and the curve by mixing Buffer A and Buffer B in a 50:1 ratio.

    5. Add 200 μl of reaction mixture to each well.

    6. Incubate at 37°C for 30 min.

    7. Measure the absorbance at 562 nm.

Data analysis

  1. Use the LibreOffice Calc software to estimate cholesterol and protein concentration in each sample by interpolating into the appropriate standard curve. These values correspond to cholesterol content in 50 μl of extract and proteins in 1 μl of lysate (considering the 10-fold dilution).

  2. Multiply cholesterol levels by 16 to obtain levels in 800 μl of chloroform and protein levels by 50 to obtain the amount in the entire lysate.

  3. Express cholesterol levels as μg of cholesterol per μg of protein to compare different samples.

  4. Compare groups in the R statistical environment. When two groups are compared, a t-test is performed; if more than two groups are being analyzed, use ANOVA with a Tukey’s post-test. In Figure 3, we compare cholesterol levels in vascular and parenchymal fractions. Since both fractions come from the same sample, a paired t-test was performed.



    Figure 3. Cholesterol content in brain fractions. Vascular and parenchymal fractions were isolated using dextran-based centrifugation, and the cholesterol content in each fraction was determined. *P < 0.05; paired t-test.

Notes

  1. E15.5 is the earliest time point at which this protocol has worked in our hands. It is likely due to the fact that the procedure relies on the high density of the endothelium-pericyte-astrocyte endfeet complex, which begins to mature at around E15.5.

  2. Since genotyping cannot be done in a short time frame, all embryos need to be processed for vascular fragment isolation, even if only some of the embryos are expected to carry useful genotypes. After genotyping, we use the samples from embryos with required genotypes without excluding any sample.

  3. The easiest way is to put the head sideways is to grab the skin just above the presumptive ear with one forceps and begin peeling the rest of the skin with the other forceps. Just below the skin and covering the brain are the translucent meningeal membranes. To remove them, pinch carefully between forebrain hemispheres with one forceps and peel away the membranes with the other (Figure 1).

  4. The tissue will look gelatinous but evenly distributed in the dextran solution. In our hands, it looks more like a homogenate than a lightly disrupted tissue.

  5. The parenchymal fraction will remain in the supernatant, whereas the vascular fragments will be pelleted. Care should be taken to recover as much parenchyma as possible.

  6. Lysis occurs rapidly after thawing the pellet in lysis buffer, but fibrous material may be present after lysis of the vascular fraction. We include this in the lipid extraction but remove it by centrifugation for other applications, such as Western blotting.

  7. This strategy for pooling pellets can be used to mix several different samples without increasing the volume; this is useful if detection methods require more material than what is possible to obtain from one embryonic brain. When collecting samples for new applications, we always test the new assay using pools of 1-3 samples to determine the detection limit. In our hands, a sample from 1 embryonic brain is sufficient for cholesterol determination and Western blotting.

  8. Polypropylene tubes may react with chloroform, especially when heated. If tubes change form, small glass tubes should be used instead. In our hands, polypropylene tubes normally withstand the conditions of this procedure, but glass tubes may be preferable.

  9. DuraSeal is a solvent-resistant parafilm replacement that will prevent solvent evaporation during heating. Three layers should be applied to tightly seal the tube by covering the tube cap with a film square and twisting and recovering twice. When done correctly, no chloroform should leak when inverting the tube.

  10. It is important to resuspend precipitated lipids quantitatively. We have used vortexing, but it is common to use a bath sonicator for 15 min. Both time and type of resuspension should be tested when setting up this procedure.

Recipes

  1. PBS 1×

    Dilute 100 ml of PBS 10× to 1 L with MilliQ water

  2. 18% Dextran

    To prepare enough to process one brain, dissolve 9 mg of dextran 70 kDa in 500 μl of PBS 1×

    Prepare enough volume to process all brains, considering an excess volume of 1 ml

  3. Lysis buffer

    50 mM Tris

    150 mM NaCl

    1% IGEPA

    0.1% Sodium Dodecyl Sulfate

    Protease inhibitors 1×

  4. Running buffer

    Mix 100 ml of Tris-Glycine 10× and 10 ml of SDS 10% and bring to 1 L with MilliQ water

  5. Transfer buffer

    Mix 100 ml of Tris-Glycine 10× and 200 ml of methanol and bring to 1 L with MilliQ water

  6. TBST 1×

    Dilute 100 ml of TBST 10× to 1 L with MilliQ water

Acknowledgments

N.S. is supported by AHA Postdoctoral fellowship 20POST35120371. This protocol is based on Boulay et al. (2015).

Competing interests

The authors declare that no competing interests exist.

Ethics

Animal procedures described here follow AVMA guidelines and have been approved by the Institutional Review Board of the University of California, San Francisco (AN177934 2019-2022).

References

  1. Armulik, A., Abramsson, A. and Betsholtz, C. (2005). Endothelial/pericyte interactions. Circ Res 97(6): 512-523.
  2. Boulay, A. C., Saubamea, B., Decleves, X. and Cohen-Salmon, M. (2015). Purification of Mouse Brain Vessels. J Vis Exp(105): e53208.
  3. Daneman, R., Zhou, L., Agalliu, D., Cahoy, J. D., Kaushal, A. and Barres, B. A. (2010). The mouse blood-brain barrier transcriptome: a new resource for understanding the development and function of brain endothelial cells. PLoS One 5(10): e13741.
  4. Gavins, F., Yilmaz, G. and Granger, D. N. (2007). The evolving paradigm for blood cell-endothelial cell interactions in the cerebral microcirculation. Microcirculation 14(7): 667-681.
  5. Santander, N., Lizama, C. O., Meky, E., McKinsey, G. L., Jung, B., Sheppard, D., Betsholtz, C. and Arnold, T. D. (2020). Lack of Flvcr2 impairs brain angiogenesis without affecting the blood-brain barrier. J Clin Invest 130(8): 4055-4068.
  6. Tan, X., Liu, W. A., Zhang, X. J., Shi, W., Ren, S. Q., Li, Z., Brown, K. N. and Shi, S. H. (2016). Vascular Influence on Ventral Telencephalic Progenitors and Neocortical Interneuron Production. Dev Cell 36(6): 624-638.
  7. Vasudevan, A., Long, J. E., Crandall, J. E., Rubenstein, J. L. and Bhide, P. G. (2008). Compartment-specific transcription factors orchestrate angiogenesis gradients in the embryonic brain. Nat Neurosci 11(4): 429-439.
  8. Yousif, S., Marie-Claire, C., Roux, F., Scherrmann, J. M. and Decleves, X. (2007). Expression of drug transporters at the blood-brain barrier using an optimized isolated rat brain microvessel strategy. Brain Res 1134(1): 1-11.
  9. Zlokovic, B. V. (2008). The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron 57(2): 178-201.

简介

[摘要]大脑中的内皮细胞与其他类型的细胞相互作用,形成血脑屏障。该屏障控制溶质进出大脑的运动,调节病理生理过程和药物向大脑的输送。Ç ommon分离方法用于吨ö研究胚胎发育过程中,这些细胞包括酶处理和使用细胞特异性标记排序。这个过程改变细胞状态并产生微量样品。在这里,我们描述了一种基于葡聚糖分离从胚胎大脑中富集血管细胞的协议。在该方法中,所述脑轻轻用杵破碎,然后再悬浮在葡聚糖溶液。低- 高速离心允许实质和血管部分的分离。进一步的离心步骤改进了分级分离。这种方法是简单和快速的和生物化学测定产生足够的样品。

图文摘要:

从血管片段的纯化的胚胎脑

[背景]大脑中的内皮细胞形成一个专门的屏障,限制和控制大分子和小分子从血流进入大脑(Zlokovic ,2008)。在他们的自然利基,脑内皮细胞相互作用密切与几个细胞类型,包括周细胞,神经胶质细胞和免疫细胞,这是不可或缺的大脑平衡(Armulik等,2005; Gavins等,2007)。脑内皮细胞生物学的研究从使用程序分离内皮或血管复合体中获益匪浅,其中包括内皮细胞、周细胞和神经胶质接触(桡神经胶质、少突胶质细胞和星形胶质细胞末端)。前者通常通过荧光激活细胞分选 (FACS) 分离实现(Daneman等人,2010),而后者通过在葡聚糖溶液中离心来实现(Yousif等人,2007)。基于 FACS 的分离可以改变内皮细胞的特性,而且昂贵且效率低下,导致分离出极少数可能无法反映其天然状态的细胞。获得源自胚胎的大脑,其中样品时,这些问题更为明显的样品极为有限,和较少的通常由一个脑获得超过5000个内皮细胞。这个数字足以使用专门的试剂盒进行转录分析 (Santander et al. , 2020),但不足以进行生化分析。相反地,血管复合物的基于葡聚糖隔离尚未应用于胚胎脑小号因为标准协议需要从单个小鼠胚胎获得一个更大的样品。从单个胚胎大脑中获取足够的样本进行生化分析的能力对于研究发育性血管疾病(例如 Fowler 综合征)是必要的(Santander等,2020)。在这里,为了克服这些样本大小问题,我们描述了基于葡聚糖的血管碎片分离范式对单个小鼠胚胎大脑的适应性。我们证明了血管碎片分离在测量胚胎脑血管中胆固醇含量方面的效用,这是一种使用 FACS 分离的内皮细胞不可行的检测方法。

关键字:胚胎, 大脑, 脉管系统, 葡萄聚糖, 胆固醇



材料和试剂


15毫升锥形底管
DuraSeal实验室薄膜(Sigma,目录号:D3172)
聚丙烯 1.5 毫升管
检测板,96 孔,黑色
分析板,96 孔,透明
PVDF膜(Thermo,目录号:88520)
培养板
雌性和雄性小鼠
PBS 10 × (Apex,目录号:20-134)
葡聚糖(70 kDa的高度吸湿的,应当在保持一个惰性气氛中)(TCI,目录号:D1449)
IGEPAL(Sigma,目录号:I8896)
十二烷基硫酸钠(Sigma,目录号:L3771)
蛋白酶抑制剂混合物(Thermo,目录号:78429)
MilliQ水
BCA蛋白质测定试剂盒(Thermo,目录号:23225)
甲醇(默克,目录号:1060091000)
氯仿(默克,目录号:1070242500)
Amplex红胆固醇测定K它(Thermo,目录号:A12216)
蛋白质加载缓冲液 4 × (Bio - Rad,目录号:1610747)
2-巯基乙醇(Sigma,目录号:M6250)
Buffer Tris-Glycine 10 × (Apex,目录号:18-238B)
预制 10% 聚丙烯酰胺凝胶(Bio-Rad,目录号:4561033)
脱脂奶粉
TBST 10 × (Apex,目录号:18-235B)
兔抗 ERG(Abcam,目录号:ab92513)
兔抗PECAM1(Santa Cruz,目录号:sc-1506)
兔抗 PAX6(Millipore,目录号:AB2237)
兔抗TBR1(Abcam,目录号:ab31940)
小鼠抗ACTINB(R&D Systems,目录号:MAB8929)
驴抗兔IGG(Thermo,目录号:A16029)
驴防鼠IGG(Thermo,目录号:A16011)
化学发光试剂盒(Bio - Rad,目录号:1705061)
PBS 1× (见食谱)
18% 葡聚糖(见食谱)
裂解缓冲液(见配方)
运行缓冲液(见配方)
转移缓冲液(见配方)
TBST 1× (见食谱)


设备


Dumont #5 精细镊子(Fine Science Tools,目录号:11251-30)
管杵(Sigma,目录号:Z359947)
CO 2安乐死室
解剖显微镜
冷藏微量离心机
加热块
通风柜
凝胶文件系统


软件


LibreOffice Calc
电阻


程序


胚胎脑解剖
以 2:1 或 1:1 的比例繁殖雌性和雄性小鼠。
每天在光照周期的第一个小时内检查雌性是否存在阴道塞。
用插头将雌性分开,以避免重新插入,并将那天早上注册为胚胎日 (E) 0.5。
在 E15.5 天,按照机构准则对女性实施安乐死。我们结合使用 CO 2窒息和颈椎脱位。见注 1。
取出子宫,将它们放入 PBS 1 × 中,并将每个植入部位分开。应注意保持卵黄囊完整,以尽量减少组织损伤。
在解剖显微镜下,解剖没有胚外组织的胚胎。如果需要进行基因分型,请切下尾巴的尖端或爪子并将其放入管中。见注 2。
使用 Dumont #5 细钳切开胚胎头并剥离覆盖大脑的皮肤和脑膜。参见注释 3 和图 1 。






图1的夹层一个小鼠胚胎大脑。使用 Dumont #5 镊子(左)沿虚线剥去头部皮肤。脑膜层小号覆盖大脑小心地移除以暴露脑(中心,虚线)。从侧面释放轻轻推大脑Ş从头部(右)的整个器官。


通过在颅骨底部切割菱脑并从侧面轻轻推动前脑,将大脑从头部取出。
将大脑放入 1 ml 冰冷 PBS 中的 1.5 ml 聚丙烯管中。


V ascular片段我溶胶化(所有步骤应在冰上进行)
使用无菌 1.5 毫升管杵用五次敲击打乱大脑。
在 4°C 下以 1,500 × g离心20 分钟。
在离心过程中,在 PBS 中准备足够的 18% 葡聚糖溶液,以单独处理收集的每个大脑。
弃去上清液,将沉淀重悬在 500 μl 18% 葡聚糖中。见注 4。
在 4°C 下以 1,500 × g离心20 分钟。
将上清液转移到另一管中。见注 5。
将沉淀重悬在 500 μl 18% 葡聚糖中。
在 4°C 下以 1,500 × g离心这两个级分20 分钟。颗粒是血管碎片。
转移的上清以一个不同的管中,加入1ml的PBS。
将稀释的上清液在4°C 下以 10,000 × g离心10 分钟,然后弃去上清液。颗粒是实质部分。
将血管和实质部分储存在 < -70°C 直至使用。


用于评估馏分富集的蛋白质印迹法
将 100 μl裂解缓冲液直接加入冰上的沉淀中。应当小心,以保持冻结直到沉淀的裂解缓冲液通过保持加入的干冰上或液氮的管。
通过上下吹打来溶解样品,直到溶解物看起来均匀。见注 6。
通过将所有裂解物从第一个管转移到第二个管,池对应于同一样品的相同部分的颗粒。通过移液溶解第二个颗粒。见注 7。
将 10 μl裂解物转移到不同的 1.5 ml 管中进行蛋白质定量。保持在 < -70°C。
对每个要平行分析的样品重复步骤 C1- C 4。始终与实验样品一起处理控制。
将 2-巯基乙醇加入上样缓冲液中,使其浓度为 5%。
将 15 μg蛋白质的等分试样与 1/3 体积的上样缓冲液与 2-巯基乙醇混合,并在 98°C 下加热 5 分钟。
加载到10%聚丙烯酰胺凝胶并运行用于在30的范围内适当的时间来解决的蛋白质kDa的和150 kDa的在运行缓冲液。
在甲醇中孵育 PVDF 膜 5 分钟以进行活化。
在转移缓冲液中以 300 mA 将蛋白质转移到活化的 PVDF 膜中 1 小时。
通过在 5% 脱脂牛奶、0.01% Tween 20 TBS 1 × 中孵育 2 小时来封闭膜。
用在封闭缓冲液中以1:2 , 000稀释的一抗孵育膜。
在 TBS 1 ×中的 0.01% Tween 20 中清洗膜3次,每次 10 分钟。
与在封闭缓冲液中以 1:10,000 稀释的二抗一起孵育。
在 TBS 1 ×中的 0.01% Tween 20 中清洗膜3次,每次 10 分钟。
混合化学发光试剂盒中的溶液并覆盖膜 1 分钟。
在凝胶文档系统中印迹多余的溶液和图像。
的代表性印迹证实了在血管级分,并在实质分数一个神经元祖标记的内皮标志物的富集示于图2 。






图 2. 血管和实质部分的富集。血管 (V) 和实质 (P) 部分中内皮 (PECAM1、ERG) 和神经元祖细胞 (PAX6、TBR1) 标记物的蛋白质印迹分析。请注意,该PAX6信号在血管片段检测,表示脑内皮细胞表达PAX6(瓦苏德万等人,2008)和/或血管粘附神经/神经胶质祖细胞(谈等人,2016)。


脂质提取
将 100 μl裂解缓冲液直接加入冰上的沉淀中。应注意保持沉淀冷冻,直到通过将管保持在干冰或液氮上来添加裂解缓冲液。
通过上下吹打来溶解样品,直到溶解物看起来均匀。见注 6。
通过将所有裂解物从第一个管转移到第二个管,池对应于同一样品的相同部分的颗粒。通过移液溶解第二个颗粒。见注 7。
将 10 μl裂解物转移到不同的 1.5 ml 管中进行蛋白质定量。保持在 < -70°C。
对每个要平行分析的样品重复步骤 D1-D4。始终与实验样品一起处理控制。
在通风橱中,向 90 μl裂解液中加入 400 μl甲醇,然后加入 800 μl氯仿。见注 8。
用DuraSeal实验室薄膜密封管。见注 9。
涡旋每管 1 分钟,使两相混合均匀。
使用加热块或浴在 60°C 下加热 30 分钟。
涡旋 1 分钟。
在 4°C 下孵育过夜以使相分离。
在 4°C 下以 1,500 × g离心20 分钟。
转移400 μl的疏水相(底层)的在至新管。为避免有机相被水相污染,在将空气引入溶液之前,将一些空气推入移液管尖端,然后将其释放到下相的上部。
将 50 μl有机相转移到新管中并在惰性气氛下蒸发。蒸发后,应看不到结晶。应以这种方式为每个样品准备两管,以重复测量胆固醇。


胆固醇测量
准备所有试剂中的Amplex红胆固醇分析ķ它,按照制造商的说明。
将 50 μl反应缓冲液 1 × 添加到含有蒸发有机相的管中。
涡旋 1 分钟。见注释 10。
转移到黑色 96 孔板。
构建以下胆固醇标准曲线的制造商的说明。将 50 μl转移到黑色 96 孔板中。
按照制造商的描述设置反应混合物,并在每个含有样品和曲线的孔中加入 50 μl 。
在 37°C 下孵育 30 分钟。
测量的与在560nm处的激发和发射在590nm的荧光。


蛋白质测量
在冰上解冻的样品等分试样和制备100微升通过添加90 10倍稀释的微升的的MilliQ水到每个管中。
按照制造商的指示,通过稀释 BSA 标准来构建 BSA 曲线。
将 25 μl的每个样品和曲线点转移到透明的 96 孔板中。
通过以 50:1 的比例混合缓冲液 A 和缓冲液 B,为所有样品和曲线准备足够的反应混合物。
向每个孔中加入 200 μl反应混合物。
在 37°C 下孵育 30 分钟。
测量562 nm 处的吸光度。


数据分析


使用所述的LibreOffice Calc软件通过内插到适当的标准曲线来估计每个样品中的胆固醇和蛋白质浓度。这些值对应于 50 μl提取物中的胆固醇含量和 1 μl裂解物中的蛋白质(考虑 10 倍稀释)。
将胆固醇水平乘以 16 以获得 800 μl氯仿中的水平,将蛋白质水平乘以 50 以获得整个裂解物中的量。
将胆固醇水平表示为每微克蛋白质的微克胆固醇,以比较不同的样品。
比较 R 统计环境中的组。两组比较时,进行t检验;如果要分析两个以上的组,请使用带有 Tukey 后检验的方差分析。在图 3 中,我们比较了血管和实质部分中的胆固醇水平。由于两个部分来自同一样本,因此进行了配对t检验。






图 3. 大脑组分中的胆固醇含量。使用基于葡聚糖的离心分离血管和实质部分,并确定每个部分中的胆固醇含量。* P < 0.05;配对t检验。


笔记


E15.5 是该协议在我们手中起作用的最早时间点。这可能是由于该过程依赖于高密度的内皮-周细胞-星形胶质细胞末端复合物,其在 E15.5 左右开始成熟。
由于基因分型无法在短时间内完成,因此所有胚胎都需要进行处理以分离血管片段,即使预计只有部分胚胎具有有用的基因型。基因分型后,我们使用了从与所需的基因型的胚胎样本不排除任何样品。
Ť他最简单的办法就是把侧着脑袋是要抢的正上方推定耳钳一个皮肤,并开始与其他镊子剥离皮肤的其余部分。就在皮肤下方并覆盖大脑的是半透明的脑膜。要移除它们,请用一个镊子小心地夹在前脑半球之间,然后用另一个镊子剥离膜(图 1 )。
组织看起来呈凝胶状,但均匀分布在葡聚糖溶液中。在我们手中,它看起来更像是匀浆,而不是轻微破碎的组织。
实质部分将保留在上清液中,而血管碎片将被沉淀。应注意尽可能多地恢复实质。
在裂解缓冲液中解冻沉淀后,裂解迅速发生,但在血管部分裂解后可能存在纤维物质。我们将其包含在脂质提取中,但通过离心将其去除以用于其他应用,例如蛋白质印迹。
这种策略用于汇集粒料可以用于在不增加混合几种不同样品的体积; 如果检测方法需要的材料多于从一个胚胎大脑中获得的材料,这将非常有用。当收集样品为新的应用,我们总是使用1个池测试新的测定法- 3个样品,以确定检测极限。在我们手中,来自 1 个胚胎大脑的样本足以用于胆固醇测定和蛋白质印迹。
聚丙烯管可能与氯仿发生反应,尤其是在加热时。如果管子改变形状,应使用小玻璃管代替。在我们手中,聚丙烯管通常能承受此过程的条件,但玻璃管可能更可取。
DuraSeal是一种耐溶剂的封口膜替代品,可防止加热过程中溶剂蒸发。Ť重稀土层应当施加吨ö紧密地密封所述覆盖管帽用薄膜方管和扭曲并回收两次。如果操作正确,倒置试管时不应泄漏氯仿。
定量重悬沉淀的脂质很重要。我们使用了涡旋,但通常使用浴式超声波仪15 分钟。设置此程序时,应测试再悬浮的时间和类型。


食谱


PBS 1 ×
用MilliQ水将100 ml PBS 10 ×稀释至 1 L


18% 葡聚糖
为了准备足够处理一个大脑,将 9 mg 葡聚糖 70 kDa溶解在 500 μl PBS 1 ×


准备足够的体积来处理所有大脑,考虑到 1 毫升的多余体积


裂解缓冲液
50 mM Tris


150 毫米氯化钠


1% 国际环保署


0.1% 十二烷基硫酸钠


蛋白酶抑制剂 1 ×


运行缓冲区
将 100 ml Tris-Glycine 10 ×和10 ml SDS 10% 混合,并用MilliQ水调至 1 L


传输缓冲区
将 100 ml Tris-Glycine 10 ×和200 ml 甲醇混合,并用MilliQ水调至 1 L


TBST 1 ×
用MilliQ水将100 ml TBST 10 ×稀释至 1 L


致谢


NS 由 AHA 博士后奖学金支持 20POST35120371。该协议基于 Boulay等人。(2015) 。


利益争夺


作者声明不存在竞争利益。


伦理


此处描述的动物程序遵循 AVMA 指南,并已获得加州大学旧金山分校机构审查委员会的批准(AN177934 2019-2022)。


参考


Armulik , A.、Abramsson , A. 和Betsholtz , C. (2005)。内皮/周细胞相互作用。Circ Res 97(6): 512-523。
Boulay, AC, Saubamea , B., Decleves , X. 和 Cohen-Salmon, M. (2015)。小鼠脑血管的纯化。J Vis Exp ( 105):e53208。
Daneman,R.,周,L.,Agalliu ,D.,Cahoy ,JD,Kaushal,A和巴赫斯,BA(2010)。小鼠血脑屏障转录组:了解脑内皮细胞发育和功能的新资源。PLoS一5(10):e13741。
Gavins , F., Yilmaz, G. 和 Granger, DN (2007)。脑微循环中血细胞-内皮细胞相互作用的进化范式。微循环14(7):667-681。
Santander, N., Lizama , CO, Meky , E., McKinsey, GL, Jung, B., Sheppard, D., Betsholtz , C. 和 Arnold, TD (2020)。缺乏 Flvcr2 会损害脑血管生成而不影响血脑屏障。J Clin Invest 130(8): 4055-4068。
Tan, X., Liu, WA, Zhang, XJ, Shi, W., Ren, SQ, Li, Z., Brown, KN 和 Shi, SH (2016)。血管对腹侧端脑祖细胞和新皮质中间神经元产生的影响。开发单元36(6):624-638。
Vasudevan, A., Long, JE, Crandall, JE, Rubenstein, JL 和Bhide , PG (2008)。室特异性转录因子协调胚胎大脑中的血管生成梯度。Nat Neurosci 11(4): 429-439。
Yousif, S., Marie-Claire, C., Roux, F., Scherrmann , JM 和Decleves , X. (2007)。使用优化的离体大鼠脑微血管策略在血脑屏障上表达药物转运蛋白。大脑研究1134(1): 1-11。
兹洛科维奇,BV(2008 年)。健康和慢性神经退行性疾病中的血脑屏障。神经元57(2):178-201。
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引用:Santander, N. and Arnold, T. D. (2021). Enrichment of Vascular Fragments from Mouse Embryonic Brains for Endothelial Cell Analysis. Bio-protocol 11(12): e4058. DOI: 10.21769/BioProtoc.4058.
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