搜索

ELISA Detection of Endogenous Serum Albumin in the Mouse Brain: A Measure of Extravasation Following Brain Injury
脑损伤后外渗的一种检测方法:酶联免疫吸附试验检测小鼠脑内血清白蛋白   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

After stroke and brain contusion, serum proteins extravasate into nerve tissue through disrupted blood-brain barrier (BBB). Because extravasations of serum proteins result in vasogenic brain edema, serum albumin level in the brain is an indicator of BBB disruption and brain edema after brain insults. In this protocol, extravasation of endogenous albumin is measured in the damaged mouse brain, which would be valuable in the evaluation of vasogenic brain edema formation (Michinaga et al., 2014).

Keywords: Blood-brain barrier(血脑障壁), Brain edema(脑水肿), Endothelial permeability(内皮通透性), Brain microvessel(脑微血管)

Materials and Reagents

  1. Mouse Albumin ELISA Quantitation Set (Bethyl Laboratories, catalog number: E90-134 )
    Note: This set includes an anti-mouse albumin goat antibody for plate coating, a mouse reference serum and a HRP-conjugated anti-mouse albumin goat antibody.
  2. Mouse serum albumin (Sigma-Aldrich, catalog number: A3139 )
  3. SureBlueTM TMB microwell peroxidase substrate (Kirkegaard & Perry Laboratories, catalog number: 52-00-01 )
  4. 450 nm BioFX® liquid Nova-Stop solution for TMB microwell substrates (SurModics, catalog number: LSTP-0100-01 )
  5. BCA protein assay reagent A (Thermo Fisher Scientific, catalog number: 23221 )
  6. BCA protein assay reagent B (Thermo Fisher Scientific, catalog number: 23224 )
  7. Bovine serum albumin (BSA) set (Thermo Fisher Scientific, catalog number: 23208)
  8. Pentobarbital sodium salt (Nacalai Tesque, catalog number: 26427-14 )
  9. PBS (Santa Cruz Biotechnology, catalog number: sc-24946 )
  10. Triton X-100 (Nacalai Tesque, catalog number: 35501-15 )
  11. Tris (hydroxymethyl) aminomethane (Nacalai Tesque, catalog number: 35409-45 )
  12. HCl (Nacalai Tesque, catalog number: 18321-05 )
  13. NaCl (Nacalai Tesque, catalog number: 31320-05 )
  14. NP-40 (Nacalai Tesque, catalog number: 23640-94 )
  15. Deoxycholic acid (Wako Chemicals USA, catalog number: 044-18812 )
  16. SDS (Nacalai Tesque, catalog number: 31607-65 )
  17. EDTA (Nacalai Tesque, catalog number: 15114-15 )
  18. Phenylmethylsulfonyl fluoride (Enzo Life Science, catalog number: ALX-270-184-G025 )
  19. Aprotinin (Sigma-Aldrich, catalog number: A6103 )
  20. PBS (see Recipes)
  21. PBST (see Recipes)
  22. Lysis buffer (see Recipes)

Equipment

  1. Scissors (Fine Science Tools, catalog number: 14002-14 )
  2. Forceps (Fine Science Tools, catalog number: 11271-30 )
  3. Hemostats (Fine Science Tools, catalog number: 13002-10 )
  4. Winged needle (Terumo, catalog number: SV-27DL )
  5. Syringe (Terumo, catalog number: SS-30ESZ )
  6. Razor (FEATHER Safety Razor, catalog number: FH-10 )
  7. Acrylic brain slicer (Muromachi Kikai, catalog number: MK-MC-01 )
  8. 1.5 ml sampling tubes (Bio-Bik, catalog number: RC-0170 )
  9. 15 ml conical tube (BD Biosciences, Falcon®, catalog number: REF 352196 )
  10. 96-well plate (Thermo Fisher Scientific, catalog number: 167008 )
  11. Pipette tips (2 to 200 µl) (Eppendorf, catalog number: 0030 073. 800 )
  12. Stereotactic device (Narishige, model: SR-6N )
  13. ELISA plate set (Sumitomo Bakelite, catalog number: BS-X7310 )
    Note: This set includes ELISA plates, plate seals, an antibody-immobilizing solution and a soaking solution.
  14. Micropipettes (2 to 20 µl and 20 to 200 µl, Eppendorf)
  15. Microplate reader (Bio-Rad Laboratories, model: 680 )
  16. Digital Sonifier® (Branson, model: 250D )
  17. Centrifuge (Hitachi, model: CF15RXII )
  18. Microplate mixer (AS ONE Corporation, model: NS-4P )

Procedure

  1. Disruption of BBB by cerebral cold injury [see Material and Methods in Michinaga et al. (2014)]
    Mice (5 to 6 week old male ddY, SLC) were placed in a stereotactic device (Narishige, model: SR-6N) under anesthesia. The scalp was incised at the midline and the skull was exposed. A copper rod (10 cm length, 4 mm diameter) was kept in a 50 ml conical tube filled with powdered dry ice. The cooled rod was set on a micromanipulator with the conical tube. The tip of the cooled rod was applied to the skull of the left hemisphere at a position of 3 mm posterior and 5 mm lateral to bregma for 60 sec. In this study, the injured mice were recovered for 72 h prior to perfusion.


    Figure 1. Procedure of cerebral cold injury. A copper rod (10 cm length, 4 mm diameter) was shown in the left photograph. It was set in a 50 ml conical tube and cooled by dry ice powder. After mice were placed in a stereotactic device under anesthesia, the cooled rod was attached in the left hemisphere on the skull shown in middle and right photographs.

  2. Mouse perfusion
    1. Make experimental manipulations to reproduce brain insults. Disruption of BBB and brain edema will gradually develop thereafter.
    2. Anesthetize mice deeply by pentobarbital (50 mg/kg, i. p.).
    3. Perform laparotomy until the heart can be clearly seen.
    4. Tear the liver for outflow of the circulating blood.
    5. Connect the wing needle and syringe.
    6. Prick the winged needle into left cardiac ventricle and slowly inflow 50 ml PBS for about 20 min checking the outflow of blood from the liver.

  3. Protein samples from the mouse cerebrum
    1. Decapitate mice after perfusion by scissors and peel the skull by forceps.
    2. Set the cerebrum in the acrylic brain slicer and cut the cerebrum in coronal brain sections (4 mm thick between 1 and 5 mm posterior to bregma) by razor (Figure 2A-C).
    3. Collect the brain tissue in sampling tubes containing 200 µl of the lysis buffer (Figure 2D).
    4. Homogenize the brain tissue by Digital Sonifier® (20 kHz, 200 W) (Figure 2E-F).
    5. Centrifuge the lysates at 15,000 x g for 10 min at 4 °C.
    6. Collect the supernatant in sampling tubes (Figure 2G).


      Figure 2. Procedure of mouse perfusion with PBS and collections of perfused cerebrum. A. Perfused mouse cerebrum was isolated and set in the acrylic brain slicer. B. The cerebrum in coronal brain section was cut. C. The injured cortex was isolated from the coronal brain section. D. Collection of the injured mouse cortex. The injured cortex was collected in sampling tubes containing 200 µl of the lysis buffer. E. Digital Sonifier® for homogenization was shown. F. The collected hemispheres were homogenized by Digital Sonifier®. G. After the homogenized samples were centrifuged, the supernatant was collected in sampling tubes.

  4. Measurement of proteins
    1. Dilute samples 10-fold by lysis buffer.
    2. Add 20 µl BSA standards (0, 125, 250, 500 and 750 µg/ml) or 20 µl samples to each well in the 96-well plate.
    3. Mix BCA Protein Assay Reagent A and BCA Protein Assay Reagent B (at rate of 50:1) in a 15 ml conical tube.
    4. Add 150 µl mixture of BCA Protein Assay Reagent A and BCA Protein Assay Reagent B to each well in the 96-well plate.
    5. Incubate at room temperature (RT) for 30 min.
    6. Measure absorbance at OD570 nm by a microplate reader. The protein samples were diluted in 0.2 mg/ml prior to ELISA.

  5. Preparation of ELISA plate
    1. Dilute anti-mouse albumin goat antibody in 1/100 with immobilizing solution.
    2. Add 100 µl of the diluted anti-mouse albumin to each well.
    3. Seal the plate and incubate it at RT for 4 h.
    4. Rinse the plate with PBST (200 µl to each well) 3 times.
    5. Add 200 µl of soaking solution to each well.
    6. Incubate at RT for 5 min.
    7. Dry the plate at RT.
    8. Pack the plate and store at 4 °C at overnight. If you want to finish the ELISA in one day, you can skip this step.

  6. ELISA
    1. Dilute mouse serum albumin at each concentration (0, 6.25, 12.5, 50 and 100 ng/ml) by lysis buffer. These mouse serum albumin solutions are used as standard solutions.
    2. Add 50 µl albumin standard solutions or 50 µl samples (10 µg protein) to each well in the prepared ELISA plate.
    3. Seal the plate and incubate it at RT for 2 h.
    4. Wash the plate with PBST (200 µl to each well) by using a microplate mixer at medium speed for 5 min 5 times.
    5. Remove PBST and dry the plate at RT for 15 min.
    6. Add 100 µl of a HRP-conjugated anti-mouse albumin goat antibody (diluted 1/1,000) with PBS to each well.
    7. Seal the plate and incubate it at RT for 1 h.
    8. Wash the plate with PBST (200 µl to each well) by using a microplate mixer at medium speed for 5 min 5 times.
    9. Add 100 µl of SureBlueTM TMB Microwell Peroxidase Substrate to each well.
    10. Incubate at RT for 5 min.
    11. Add 100 µl of 450 nm BioFX® Liquid Nova-Stop Solution for TMB Microwell Substrates to each well.
    12. Read the plate using a microplate reader at 450 nm.
    13. Calculate the concentrations of albumin using the standard curve.

Representative data

  1. Albumin contents in the mouse cerebrum after cold injury (with PBS perfusion).

    Table 1. Concentrations of albumin standard solutions and their OD450 values. Absorbance of albumin standard solutions was measured at 450 nm. The Albumin standards were duplicated and the averages of duplicated standards were shown.



    Figure 3. Standard curve of albumin solutions. The standard curve was designed from the OD450 values and the concentrations of albumin standard summarized in Table 1. The vertical axis shows the value of OD450 and the horizontal axis shows albumin standard content.

    Table 2. Albumin contents in the perfused mouse cerebrum after cold injury. After cold injury, mice were perfused with PBS. The albumin contents (ng/ml) in mice cerebrum were calculated by equation from the standard curve shown in Figure 3. Brain tissue extracts were diluted at 0.2 mg/ml. Albumin/brain tissue protein (ng/mg) were calculated by dividing albumin contents (ng/ml) by brain tissue protein contents (0.2 mg/ml).


  2. Albumin contents in the mouse cerebrum after cold injury (without PBS perfusion).

    Table 3. Concentrations of albumin standard solutions and their OD450 values. As with Table 1, absorbance of albumin standard solutions was measured at 450 nm.


    Figure 4. Standard curve of albumin solutions. The standard curve was designed from data in Table 3. The vertical axis shows the OD450 value and the horizontal axis shows albumin standard contents.



    Table 4. Albumin contents in the perfused mouse cerebrum after cold injury (without PBS perfusion). After cold injury, mice cerebrums were collected. The albumin contents (ng/ml) in mice cerebrum were calculated by equation from the standard curve shown in Figure 4. Brain tissue extracts were diluted at 0.2 mg/ml.  Albumin/brain tissue protein (ng/mg) were calculated by dividing albumin contents (ng/ml) by brain tissue protein contents (0.2 mg/ml).

Notes

  1. In non-injured brain, extravasations of serum proteins are rarely observed. As is indicated above, brain albumin contents of non-injured mouse were reduced to very low levels by perfusion with PBS. This suggests an importance of a successful washing-out of blood components in brain vessels.

Recipes

  1. PBS
    Dilute 10x PBS in distilled water
  2. PBST
    PBS
    0.05 % Triton X-100
  3. Lysis buffer
    20 mM Tris/HCl (pH 7.4)
    Dissolve Tris (hydroxymethyl) aminomethane in distilled water and regulate pH by HCl
    150 mM NaCl
    1% (v/v) NP-40
    0.5% (v/v) deoxycholic acid
    0.1% (w/v) SDS
    0.5% (w/v) EDTA
    2 mM phenylmethylsulfonyl fluoride
    10 µg/ml aprotinin

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research (C) from the JPSP (21590108).

References

  1. Michinaga, S., Nagase, M., Matsuyama, E., Yamanaka, D., Seno, N., Fuka, M., Yamamoto, Y. and Koyama, Y. (2014). Amelioration of cold injury-induced cortical brain edema formation by selective endothelin ETB receptor antagonists in mice. PLoS One 9(7): e102009.

简介

在中风和脑挫伤后,血清蛋白通过破裂的血脑屏障(BBB)外渗到神经组织中。 因为血清蛋白的外渗导致血管性脑水肿,脑中的血清白蛋白水平是脑损伤后BBB破坏和脑水肿的指标。 在该方案中,在损伤的小鼠脑中测量内源性白蛋白的外渗,这在评价血管性脑水肿形成中是有价值的(Michinaga等人,2014)。

关键字:血脑障壁, 脑水肿, 内皮通透性, 脑微血管

材料和试剂

  1. 小鼠白蛋白ELISA定量集(Bethyl Laboratories,目录号:E90-134)
    注意:此组包括用于包被的抗小鼠白蛋白山羊抗体,小鼠参考血清和HRP缀合的抗小鼠白蛋白山羊抗体。
  2. 小鼠血清白蛋白(Sigma-Aldrich,目录号:A3139)
  3. SureBlue TM TMB微孔过氧化物酶底物(Kirkegaard& Perry Laboratories,目录号:52-00-01)
  4. 用于TMB微孔底物(SurModics,目录号:LSTP-0100-01)的450nm BioFX?液体Nova-Stop溶液
  5. BCA蛋白测定试剂A(Thermo Fisher Scientific,目录号:23221)
  6. BCA蛋白测定试剂B(Thermo Fisher Scientific,目录号:23224)
  7. 牛血清白蛋白(BSA)组(Thermo Fisher Scientific,目录号:23208)
  8. 戊巴比妥钠盐(Nacalai Tesque,目录号:26427-14)
  9. PBS(Santa Cruz Biotechnology,目录号:sc-24946)
  10. Triton X-100(Nacalai Tesque,目录号:35501-15)
  11. 三(羟甲基)氨基甲烷(Nacalai Tesque,目录号:35409-45)
  12. HCl(Nacalai Tesque,目录号:18321-05)
  13. NaCl(Nacalai Tesque,目录号:31320-05)
  14. NP-40(Nacalai Tesque,目录号:23640-94)
  15. 脱氧胆酸(Wako Chemicals USA,目录号:044-18812)
  16. SDS(Nacalai Tesque,目录号:31607-65)
  17. EDTA(Nacalai Tesque,目录号:15114-15)
  18. 苯甲基磺酰氟(Enzo Life Science,目录号:ALX-270-184-G025)
  19. 抑酶肽(Sigma-Aldrich,目录号:A6103)
  20. PBS(请参阅配方)
  21. PBST(参见配方)
  22. 裂解缓冲液(见配方)

设备

  1. 剪刀(Fine Science Tools,目录号:14002-14)
  2. 镊子(Fine Science Tools,目录号:11271-30)
  3. Hemostats(Fine Science Tools,目录号:13002-10)
  4. 翼针(Terumo,目录号:SV-27DL)
  5. 注射器(Terumo,目录号:SS-30ESZ)
  6. Razor(FEATHER安全剃刀,目录号:FH-10)
  7. 丙烯酸脑切片机(Muromachi Kikai,目录号:MK-MC-01)
  8. 1.5ml取样管(Bio-Bik,目录号:RC-0170)
  9. 15ml锥形管(BD Biosciences,Falcon ,目录号:REF352196)
  10. 96孔板(Thermo Fisher Scientific,目录号:167008)
  11. 移液管吸头(2至200μl)(Eppendorf,目录号:0030 073.800)
  12. 立体定位装置(Narishige,型号:SR-6N)
  13. ELISA板组(Sumitomo Bakelite,目录号:BS-X7310)
    注意:该套件包括ELISA板,板密封,抗体固定溶液和浸泡溶液。
  14. 微量移液管(2至20μl和20至200μl,Eppendorf)
  15. 酶标仪(Bio-Rad Laboratories,型号:680)
  16. 数字超声波®(Branson,型号:250D)
  17. 离心机(Hitachi,型号:CF15RXII)
  18. 微孔板混合器(AS ONE Corporation,型号:NS-4P)

程序

  1. 通过大脑冷损伤破坏BBB [参见Michinaga等人的材料和方法(2014)]
    在麻醉下将小鼠(5至6周龄的雄性ddY,SLC)置于立体定位装置(Narishige,型号:SR-6N)中。头皮在中线切开,头骨暴露。将铜棒(10cm长,4mm直径)保持在装有粉末干冰的50ml锥形管中。将冷却的棒设置在具有锥形管的显微操作器上。将冷却的棒的尖端应用于左半球的颅骨,在前囟后3mm和前囱侧面5mm的位置60秒。在这项研究中,损伤的小鼠回收72 h之前灌注

    图1。 大脑寒冷伤害的程序。左侧照片中显示了铜杆(10厘米长,4毫米直径)。将其置于50ml锥形管中并用干冰粉末冷却。在麻醉下将小鼠置于立体定位装置中后,将冷却的棒连接在左半球上的中间和右照片所示的颅骨上。

  2. 鼠标灌注
    1. 进行实验操作,以重现大脑侮辱。中断 的BBB和脑水肿将逐渐发展。
    2. 通过戊巴比妥麻醉小鼠(50mg/kg,腹腔注射)
    3. 执行剖腹术,直到心脏清晰可见。
    4. 撕开肝脏流出循环血液。
    5. 连接机翼针和注射器。
    6. 将有翼的针刺入左心室,慢慢地 流入50 ml PBS约20分钟,检查血液的流出   肝。

  3. 来自小鼠大脑的蛋白质样品
    1. 用剪刀灌注后摘除小鼠,用镊子剥离头骨。
    2. 设置大脑在丙烯酸的大脑切片机和切大脑   冠状脑切片(4mm厚,1〜5mm后) 前囟石)(图2A-C)
    3. 收集脑组织在含有200μl裂解缓冲液的取样管(图2D)。
    4. 通过Digital Sonifier(20kHz,200W)(图2E-F)均质化脑组织。
    5. 将裂解物在15,000×g离心10分钟,在4℃
    6. 收集上清液在取样管(图2G)

      图2.使用PBS和灌注的大脑的集合的小鼠灌注的程序。 A。分离灌注的小鼠大脑并置于丙烯酸脑切片机中。 B.切割冠状脑切片中的大脑。 C.从冠状脑切片分离损伤的皮质。 D.损伤的小鼠皮质的收集。将损伤的皮质收集在含有200μl裂解缓冲液的取样管中。 E.显示了用于均质化的数字超声波仪®。 F.将收集的半球通过Digital Sonifier匀浆。将匀浆的样品离心后,将上清液收集在取样管中。

  4. 蛋白质测量
    1. 通过裂解缓冲液稀释样品10倍
    2. 向96孔板的每个孔中加入20μlBSA标准品(0,125,250,500和750μg/ml)或20μl样品。
    3. 在15ml锥形管中混合BCA蛋白测定试剂A和BCA蛋白测定试剂B(比率为50:1)。
    4. 向96孔板的每个孔中加入150μlBCA蛋白测定试剂A和BCA蛋白测定试剂B的混合物。
    5. 在室温(RT)下孵育30分钟
    6. 通过酶标仪测量OD 570nm处的吸光度。 在ELISA之前将蛋白质样品稀释为0.2mg/ml。

  5. ELISA板的制备
    1. 用1/100稀释抗小鼠白蛋白山羊抗体与固定溶液
    2. 向每个孔中加入100μl稀释的抗小鼠白蛋白。
    3. 密封板,并在室温下孵育4小时
    4. 用PBST冲洗板(每孔200μl)3次。
    5. 向每个孔中加入200μl浸泡溶液。
    6. 在室温下孵育5分钟。
    7. 在RT下干燥板。
    8. 包装平板并在4°C下储存过夜。 如果您想在一天内完成ELISA,您可以跳过此步骤。

  6. ELISA
    1. 稀释每种浓度的小鼠血清白蛋白(0,6.25,12.5,50和 100ng/ml)。 使用这些小鼠血清白蛋白溶液   作为标准解决方案。
    2. 在室温下孵育5分钟。
    3. 在RT下干燥板。
    4. 包装平板并在4°C下储存过夜。 如果您想在一天内完成ELISA,您可以跳过此步骤。

  7. ELISA
    1. 稀释每种浓度的小鼠血清白蛋白(0,6.25,12.5,50和 100ng/ml)。 使用这些小鼠血清白蛋白溶液   作为标准解决方案。
      ...
    2. Wash the plate with PBST (200 µl to each well) by using a microplate mixer at medium speed for 5 min 5 times.
    3. Add 100 µl of SureBlueTM TMB Microwell Peroxidase Substrate to each well.
    4. Incubate at RT for 5 min.
    5. Add 100 µl of 450 nm BioFX® Liquid Nova-Stop Solution for TMB Microwell Substrates to each well.
    6. Read the plate using a microplate reader at 450 nm.
    7. Calculate the concentrations of albumin using the standard curve.

Representative data

  1. Albumin contents in the mouse cerebrum after cold injury (with PBS perfusion).

    Table 1. Concentrations of albumin standard solutions and their OD450 values. Absorbance of albumin standard solutions was measured at 450 nm. The Albumin standards were duplicated and the averages of duplicated standards were shown.



    Figure 3. Standard curve of albumin solutions. The standard curve was designed from the OD450 values and the concentrations of albumin standard summarized in Table 1. The vertical axis shows the value of OD450 and the horizontal axis shows albumin standard content.

    Table 2. Albumin contents in the perfused mouse cerebrum after cold injury. After cold injury, mice were perfused with PBS. The albumin contents (ng/ml) in mice cerebrum were calculated by equation from the standard curve shown in Figure 3. Brain tissue extracts were diluted at 0.2 mg/ml. Albumin/brain tissue protein (ng/mg) were calculated by dividing albumin contents (ng/ml) by brain tissue protein contents (0.2 mg/ml).


  2. 冷损伤后的小鼠大脑中的白蛋白含量(不含PBS灌注)
    表3.白蛋白标准溶液的浓度及其OD 450 值。与表1一样,在450nm测量标准溶液

    图4.白蛋白 溶液的标准曲线。标准曲线由表3中的数据设计。纵轴显示OD450值,横轴显示白蛋白标准含量。



    表4.冷损伤(无PBS灌注)后灌注的小鼠大脑中的白蛋白含量。在冷损伤后,收集小鼠大脑。通过方程从图4所示的标准曲线计算小鼠大脑中的白蛋白含量(ng/ml)。将脑组织提取物以0.2mg/ml稀释。通过用白蛋白含量(ng/ml)除以脑组织蛋白质含量(0.2mg/ml)计算白蛋白/脑组织蛋白质(ng/mg)。

笔记

  1. 在非受伤脑中,很少观察到血清蛋白的外渗。 如上所述,通过用PBS灌注,非损伤小鼠的脑白蛋白含量降低到非常低的水平。 这表明成功洗出脑血管中的血液成分的重要性。

食谱

  1. PBS
    用蒸馏水稀释10x PBS
  2. PBST
    PBS
    0.05%Triton X-100
  3. 裂解缓冲液
    20mM Tris/HCl(pH7.4) 将三(羟甲基)氨基甲烷溶解在蒸馏水中,用HCl调节pH值
    150mM NaCl 1%(v/v)NP-40
    0.5%(v/v)脱氧胆酸 0.1%(w/v)SDS
    0.5%(w/v)EDTA
    2mM苯甲基磺酰氟 10μg/ml抑肽酶

致谢

这项工作得到了来自JPSP(21590108)的科学研究助理(C)的支持。

参考文献

  1. Michinaga,S.,Nagase,M.,Matsuyama,E.,Yamanaka,D.,Seno,N.,Fuka,M.,Yamamoto,Y.and Koyama,Y。 通过选择性内皮素ET亚型改善冷损伤诱导的皮质脑水肿形成 > receptor antagonists in mice。 PLoS One 9(7):e102009。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Michinaga, S. and Koyama, Y. (2015). ELISA Detection of Endogenous Serum Albumin in the Mouse Brain: A Measure of Extravasation Following Brain Injury. Bio-protocol 5(9): e1469. DOI: 10.21769/BioProtoc.1469.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。