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Inositol triphosphate (IP3) is an important second messenger that participates in signal transduction pathways in diverse cell types including hippocampal neurons. Stimulation of phospholipase C in response to various stimuli (hormones, growth factors, neurotransmitters, neurotrophins, neuromodulators, odorants, light, etc) results in hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2), a phospholipid that is located in the plasma membrane, and leads to the production of IP3 and diacylglycerol. Binding of IP3 to the IP3 receptor (IP3R) induces Ca2+ release from intracellular stores and enables the initiation of intracellular Ca2+-dependent signaling. Here we describe a procedure for the measurement of cellular IP3 levels in tissue homogenates prepared from rat hippocampal slices.

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Measurement of Inositol Triphosphate Levels from Rat Hippocampal Slices
大鼠海马脑片中肌醇三磷酸含量测定

神经科学 > 细胞机理 > 胞内信号传导
作者: Nino Tabatadze
Nino TabatadzeAffiliation: Department of Neurobiology, Northwestern University, Evanston, USA
For correspondence: n-tabatadze@northwestern.edu
Bio-protocol author page: a3041
 and Catherine Woolley
Catherine WoolleyAffiliation: Department of Neurobiology, Northwestern University, Evanston, USA
Bio-protocol author page: a3042
Vol 6, Iss 7, 4/5/2016, 1292 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1780

[Abstract] Inositol triphosphate (IP3) is an important second messenger that participates in signal transduction pathways in diverse cell types including hippocampal neurons. Stimulation of phospholipase C in response to various stimuli (hormones, growth factors, neurotransmitters, neurotrophins, neuromodulators, odorants, light, etc) results in hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2), a phospholipid that is located in the plasma membrane, and leads to the production of IP3 and diacylglycerol. Binding of IP3 to the IP3 receptor (IP3R) induces Ca2+ release from intracellular stores and enables the initiation of intracellular Ca2+-dependent signaling. Here we describe a procedure for the measurement of cellular IP3 levels in tissue homogenates prepared from rat hippocampal slices.
Keywords: Hippocampus(海马), Polarization assay(偏振法), Inositol triphosphate(三磷酸肌醇)

[Abstract]

Materials and Reagents

  1. Syringes (1 ml and 60 ml) and needles (24 and 27 gauge) (Becton Dickinson, catalog numbers: 309659 and 309653 )

  2. Single edge razor blade (IDL TechniEdge, catalog number: 05-091C )

  3. Double edge razor blade (Electron Microscopy Sciences, catalog number: 72002-10 )
  4. Costar Netwell Insert with 440 μm polyester mesh (Corning, catalog number: 3478 )

  5. Corning assay plate, black 384 well with non-binding surface (Corning, catalog number: 3575 )

  6. Conical tube (15 ml) (BD Biosciences, catalog number: REF352196 )

    Note: Currently, it is “Thermo Fisher Scientific, FalconTM, catalog number: REF352196”.
  7. Micro-centrifuge tubes (1.5 ml) (VWR International, catalog number: 20170-038 )

  8. Aerosol barrier pipette tips (30 μl and 200 μl) (Thermo Fisher Scientific, catalog numbers: 02-707-33 and 02-707-42 )

  9. Disposable transfer pipette (Thermo Fisher Scientific, catalog number: 13-711-9AM )

  10. Glass plate (Electron Microscopy Sciences, catalog number: 70312-22 )

  11. Glass staining dish (Newcomer supply, catalog number: 6816A )

  12. Coarse fritted glass bubbler (Corning, catalog number: 39533-12EC )
  13. Integrid Petri dishes (Becton Dickinson, Falcon, catalog number: 351112 )

    Note: Currently, it is “Thermo Fisher Scientific, FalconTM, catalog number: 351112 ”.
  14. Adult (47-57 days old) male and female Sprague Dawley rats (Harlan Laboratories)
  15. Hit-Hunter IP3 fluorescence polarization assay kit (DiscoveRx Corporation, catalog number: 90-0037 )
  16. 70% Perchloric acid (HClO4) (Sigma-Aldrich, catalog number: 244252 )

  17. Complete protease inhibitor cocktail tablets (Roche Diagnostics, catalog number: 11697498001 )

  18. Quick Start Bovine Serum Albumin (BSA) standard (Bio-Rad Laboratories, catalog number: 500-0206 )
  19. Protein assay reagent (Bio-Rad Laboratories, catalog number: 500-0006 )
  20. Sodium pentobarbital solution (Virbac, catalog number: 710101 )
  21. Calcium chloride solution (CaCl2) (Sigma-Aldrich, catalog number: 21115 )

  22. Magnesium chloride solution (MgCl2) (Sigma-Aldrich, catalog number: M1028 )

  23. Super glue (Super Glue Corporation, catalog number: SGP3 )

  24. 70% ethanol (Thermo Fisher Scientific, Decon labsTM, catalog number: 04-355-305 )
  25. 95% O2/5% CO2 gas mixture (Airgas, catalog number: X02OX95C2003102 )
  26. NaCl 

  27. NaHCO3
  28. Dextrose
  29. KCl
  30. NaH2PO4
  31. HEPES (pH 7.4)

  32. EGTA

  33. Sodium orthovanadate
  34. NaF
  35. Sodium pyrophosphate
  36. PMSF
  37. Artificial cerebrospinal fluid (aCSF) (see Recipes)
  38. Sucrose aCSF (s-aCSF) (see Recipes)

  39. HEPES homogenization buffer (see Recipes)


Equipment

  1. Scissors (Fine science tools, catalog numbers: 14000-12 and 14060-10 )
  2. Forceps (Stoelting, catalog numbers: 52104-37 and 52104-35 )

  3. Hemostat (Fine science tools, catalog number: 13008-12 )

  4. Spatula (Thermo Fisher Scientific, catalog number: S50822 )
  5. Accuspin micro 17R microcentrifuge (Thermo Fisher Scientific, catalog number: 13-100-676 )

  6. Biophotometer (Eppendorf AG, catalog number: 6133000010 )

  7. Leica VT1200S vibrating blade microtome (Leica Biosystems Nussloch GmbH, catalog number: 1491200S001 )

  8. Isotemp 105 water bath (Thermo Fisher Scientific, catalog number: S63077Q )

  9. Qsonica Q500 sonicator (Thermo Fisher Scientific, catalog number: 15-338-282 )

  10. Synergy 4 multimode microplate reader (BioTek Instruments)

  11. Vapor pressure osmometer 5520 (Wescor)

  12. Accumet AB15 plus pH meter (Thermo Fisher Scientific, catalog number: 13-636-AB15PC )
  13. Rotator (Labline Instruments)

  14. Micropipettes (2-20 μl, 20-200 μl and 100-1,000 μl) (Capitol Scientific, Eppendorf, catalog numbers: 3120000038 , 3120000054 and 3120000062 )


Procedure

  1. Preparation and treatment of transverse hippocampal slices
    1. Before starting the procedure
      1. Place ~ 200 ml s-aCSF per rat at -80 °C for approximately 40 min to form a slush (if s-aCSF is pre-chilled at 4 °C slush will be formed more quickly, in 15-20 min).
      2. Place Netwell insert into a glass staining dish with 350 ml regular aCSF (constantly oxygenating with 95% O2/5% CO2 mixture using a coarse glass bubbler) and put the dish into a water bath at 34-35 °C 20 min prior to slice preparation.
      3. Pour 20-30 ml s-aCSF slush in a small glass beaker, place the beaker on ice and oxygenate with 95% O2/5% CO2 mixture using a coarse glass bubbler for 10-15 min.
      4. Pre-chill the microtome stage by placing ice in the ice chamber and attach the double edge razor blade to the blade holder (Figure 2A).
      5. Bend the tip of a 27 gauge needle (~45 degrees) using forceps and place it on a 1 ml syringe.
    2. Deeply anesthetize rats with sodium pentobarbital (125 mg/kg, i.p.) and perform laparotomy using surgical scissors and forceps until the heart is completely exposed. Clamp the tip of the sternum with a hemostat and place the hemostat over the head. 

    3. Perfuse transcardially with ice-cold oxygenated (95% O2/5% CO2) s-aCSF (~50 ml/rat).
      Note: Estimated time to carry out step A3 is approximately 25 sec. 

    4. Remove the head immediately, quickly make an incision in the middle of the scalp, pull aside the skin and cut through the skull along the midline suture of the parietal plates. Remove the skull plates, carefully extract the brain using a spatula and transfer it into oxygenated and pre-chilled s-aCSF prepared in the step A1c for about one minute.
      Note: Estimated time to carry out step A4 is less than a minute.
    5. Place the brain on a glass plate on ice (Figure 1A). Hemisect the brain along the longitudinal fissure (Figure 1A, dashed line 1) and make two 45 degree angle cuts relative to the coronal plane (Figure 1A, dashed lines 2 and 3) for each hemisphere, producing tissue blocks containing the hippocampus (Figure 1B).


      Figure 1. A. Perfused rat brain on a glass plate on ice. The dashed lines indicate where cuts are made and numbers indicate the order of the cuts. B. Blocked brain with the two hemispheres separated. C. One hemisphere of the brain mounted on the mounting disk of a Leica VT1200S vibrating microtome.

    6. Place a small drop of super glue in the center of the mounting disk, carefully place one hemisphere on the drop of glue with rostral side up (Figure 1C) and transfer to the sectioning stage of the vibrating microtome (Figure 2A).


      Figure 2. A. Leica VT1200S vibrating blade microtome with stage pre-chilled with ice. B. Two transverse slices (300 μm) that were cut using the microtome, and the remaining tissue block.

    7. Immediately cover the tissue with s-aCSF slush and cut 300 μm sections (Figure 2B).
    8. Transfer the sections into separate wells of the Netwell insert (Figure 3A) using a disposable transfer pipette and let the sections recover at 34-35 °C in oxygenated regular aCSF for 30-60 min. Make sure the sections are resting flat on the mesh of each insert and are not floating or moving.


      Figure 3. A. Slice recovery and treatment set-up. B. A single slice is shown on the Integrid Petri dish before dissection of the hippocampus. Hippocampus is outlined by a dashed line in the middle of the slice.

    9. After recovery, the slices are ready for experimental treatment. Treat slices in oxygenated regular aCSF either with vehicle or a pharmacological agent of interest. Treatment times should be determined empirically (seconds to minutes). Slices from each animal should be distributed evenly between vehicle and treatment groups. We pool four slices for each sample, which typically yields 300-400 μg total protein per sample.
    10. After treatment, using a disposable transfer pipette, immediately transfer slices into the Integrid Petri dish with ~20 ml of 0.2 N perchloric acid solution made from 70% perchloric acid in regular aCSF to stabilize IP3 and incubate at room temp for 2 min. Quickly replace perchloric acid-aCSF with regular aCSF and place the Integrid Petri dish on ice.
    11. Quickly dissect hippocampi from each slice in regular aCSF on ice (Figure 3B) using a 1 ml syringe with a bent needle and, using the needle tip, quickly transfer each hippocampus into a 1.5 ml microcentrifuge tube with 300 μl ice cold HEPES homogenization buffer containing protease inhibitors (1 tablet per 25 ml buffer).

  2. Tissue homogenization and protein quantification
    1. Homogenize the tissue on ice using a sonicator (2 times, 3 sec pulses, using the 40% amplitude setting), keeping the microcentrifuge tube steady and the sonicator tip submerged half way into the liquid in order to avoid bubbling. 

    2. Centrifuge the tissue homogenates [3,000 rpm (1,000 x g) at 4 °C for 10 min] to remove unbroken cells and nuclei. Discard the pellet. 

    3. Measure protein concentration in each sample using a Bradford assay. Briefly, prepare standards with known BSA (1 mg/ml stock) concentrations and unknown samples in double distilled water. Add Bradford protein assay reagent and incubate at room temp for 5 min. Measure absorbance at 595 nm using a spectrophotometer. Build a standard curve using absorbance values from the standards and determine protein concentration for each sample based on the standard curve. 

    4. Adjust protein concentration in each sample to the same value (e.g. 2 μg/μl) using ice cold HEPES homogenization buffer with protease inhibitors. 


  3. Measuring IP3 levels
    1. Spray down the bench surface with 70% ethanol and clean it thoroughly. Make sure to carry out the IP3 assay in a dust free environment.
    2. Thaw the IP3 assay kit reagents completely and equilibrate to room temp before use.
    3. Dilute the IP3 standard (20 μM stock) to prepare ten 3-fold serial dilutions using the IP3 Standard Dilution Buffer. For the blank, use the IP3 Standard Dilution Buffer.
    4. Pipette 10 μl of the blank, standards, and each sample (20 μg protein per sample) in triplicate into wells of the 384-well assay plate with non-binding surface using barrier tips.
    5. Add 10 μl IP3 tracer to each well, followed by 20 μl IP3 binding protein and mix well by pipetting up and down several times.
    6. Carefully examine the plate for bubbles. If bubbles form, pop them with barrier pipette tips. Avoid cross-contamination by using new tip for each well. Incubate the plate for 30 min at room temp in the dark on a rotator with gentle mixing (~60 rpm).
    7. Read the fluorescence polarization signal of the IP3 tracer with a multimode microplate reader and a fluorescence polarization filter, using 485 nm excitation and 530 nm emission wavelengths.
    8. Plot a standard curve and calculate the IP3 concentration in each sample (nM range in the hippocampus). In addition to absolute concentration values, IP3 levels for each vehicle and experimental group can also be reported relative to baseline (no treatment control).

Recipes

  1. Artificial cerebrospinal fluid (aCSF, should be made fresh)
    126 mM NaCl 

    26 mM NaHCO3
    10 mM dextrose

    3 mM KCl

    1.25 mM NaH2PO4
    2 mM CaCl2
    1 mM MgCl2

    Adjust pH to 7.4 using either 2 N HCl or 5 N NaOH
    Measure osmolarity (300-310 mOsm)
  2. Sucrose aCSF (s-aCSF, should be made fresh)
    75 mM sucrose

    75 mM NaCl

    25 mM NaHCO3
    15 mM dextrose

    2.4 mM sodium pyruvate
    2 mM KCl

    1.3 mM ascorbic acid
    1.25 mM NaH2PO4
    3 mM MgCl2
    0.5 mM CaCl2
    Adjust pH to 7.4 using either 2 N HCl or 5 N NaOH

  3. HEPES homogenization buffer (can be stored at 4 °C up to three months)
    4 mM HEPES (pH 7.4)

    320 mM sucrose

    2 mM EGTA

    1 mM sodium orthovanadate
    50 mM NaF
    10 mM sodium pyrophosphate
    20 mM glycerophosphate

    0.1 mM PMSF
    Add protease inhibitor cocktail just before use in 15 ml conical tube

Acknowledgments

This research was supported by National Institutes of Health Grant R01 NS037324, the Office for Research on Women's Health, and the Northwestern University High Throughput Analysis Facility.

References

  1. Tabatadze, N., Huang, G., May, R. M., Jain, A. and Woolley, C. S. (2015). Sex differences in molecular signaling at inhibitory synapses in the hippocampus. J Neurosci 35(32): 11252-11265.

材料和试剂

  1. 注射器(1ml和60ml)和针(24和27号)(Becton Dickinson,目录号:309659和309653)
  2. 单刃剃刀刀片(IDL TechniEdge,目录号:05-091C)
  3. 双刃剃刀刀片(Electron Microscopy Sciences,目录号:72002-10)
  4. Costar Netwell插入件,带440μm聚酯网(Corning,目录号:3478)
  5. Corning测定板,具有非结合表面的黑色384孔(Corning,目录号:3575)
  6. 锥形管(15ml)(BD Biosciences,目录号:REF352196)
    注意:目前,"Thermo Fisher Scientific,Falcon ,目录号:REF352196" />
  7. 微量离心管(1.5ml)(VWR International,目录号:20170-038)
  8. 气溶胶屏障移液管吸头(30μl和200μl)(Thermo Fisher Scientific,目录号:02-707-33和02-707-42)
  9. 一次性转移移液管(Thermo Fisher Scientific,目录号:13-711-9AM)
  10. 玻璃板(Electron Microscopy Sciences,目录号:70312-22)
  11. 玻璃染色皿(Newcomer supply,目录号:6816A)
  12. 粗烧玻璃起泡器(Corning,目录号:39533-12EC)
  13. 整合培养皿(Becton Dickinson,Falcon,目录号:351112) />
  14. 成年(47-57天龄)雄性和雌性Sprague Dawley大鼠(Harlan Laboratories)
  15. Hit-Hunter IP Sub 3荧光偏振测定试剂盒(DiscoveRx公司,目录号:90-0037)
  16. 70%高氯酸(HClO 4)(Sigma-Aldrich,目录号:244252)
  17. 完全蛋白酶抑制剂混合物片剂(Roche Diagnostics,目录号:11697498001)
  18. 快速起始牛血清白蛋白(BSA)标准品(Bio-Rad Laboratories,目录号:500-0206)
  19. 蛋白质测定试剂(Bio-Rad Laboratories,目录号:500-0006)
  20. 戊巴比妥钠溶液(Virbac,目录号:710101)
  21. 氯化钙溶液(CaCl 2)(Sigma-Aldrich,目录号:21115)
  22. 氯化镁溶液(MgCl 2)(Sigma-Aldrich,目录号:M1028)
  23. 超级胶(Super Glue Corporation,目录号:SGP3)
  24. 70%乙醇(Thermo Fisher Scientific,Decon labs TM,目录号:04-355-305)
  25. 95%O 2/5%CO 2气体混合物(Airgas,目录号:XO2OX95C2003102)
  26. NaCl
  27. NaHCO 3
  28. 葡萄糖
  29. KCl
  30. NaH 2 PO 4 sub
  31. HEPES(pH 7.4)
  32. EGTA
  33. 原钒酸钠
  34. NaF
  35. 焦磷酸钠
  36. PMSF
  37. 人工脑脊液(aCSF)(参见配方)
  38. 蔗糖aCSF(s-aCSF)(参见配方)
  39. HEPES匀浆缓冲液(见配方)

设备

  1. 剪刀(精细科学工具,目录号:14000-12和14060-10)
  2. 镊子(Stoelting,目录号:52104-37和52104-35)
  3. Hemostat(精细科学工具,目录号:13008-12)
  4. 刮刀(Thermo Fisher Scientific,目录号:S50822)
  5. Accuspin micro 17R微量离心机(Thermo Fisher Scientific,目录号:13-100-676)
  6. 生物光度计(Eppendorf AG,目录号:6133000010)
  7. Leica VT1200S振动刀片切片机(Leica Biosystems Nussloch GmbH,目录号:1491200S001)
  8. Isotemp 105水浴(Thermo Fisher Scientific,目录号:S63077Q)
  9. Qsonica Q500超声波仪(Thermo Fisher Scientific,目录号:15-338-282)
  10. Synergy 4多模微板读数器(BioTek Instruments)
  11. 蒸汽压渗透压计5520(Wescor)
  12. Accumet AB15 plus pH计(Thermo Fisher Scientific,目录号:13-636-AB15PC)
  13. 旋转器(Labline Instruments)
  14. 微量移液器(2-20μl,20-200μl和100-1,000μl)(Capitol Scientific,Eppendorf,目录号:3120000038,3120000054和3120000062)

程序

  1. 横向海马切片的制备和治疗
    1. 开始操作之前
      1. 每个大鼠放置?200 ml s-aCSF -80℃下约40分钟以形成浆状物(如果s-aCSF为 预冷在4℃下形成的泥浆将更快,在15-20分钟内)。
      2. 将网室插入玻璃染色皿350毫升规则 aCSF(用95%O 2/5%CO 2混合物连续氧化) 玻璃鼓泡器),并将盘放入34-35℃的水浴中20分钟 在切片准备之前。
      3. 倒入20-30 ml s-aCSF浆液 小玻璃烧杯中,将烧杯置于冰上并用95%O 2/5% ?CO 2混合物,使用粗玻璃起泡器10-15分钟。
      4. 通过将冰放在冰室中预冷切片机台 将双刃剃须刀刀片连接到刀片架(图2A)。
      5. 使用镊子弯曲27号针(约45度)的尖端,并将其放置在1ml??注射器。
    2. 用戊巴比妥钠(125mg/kg,i.p。)深度麻醉大鼠, 并使用手术剪刀和镊子进行剖腹术直到 心脏是完全暴露的。用a夹住胸骨的尖端 止血并将止血钳放在头上。
    3. 用冰冷的含氧(95%O 2/5%CO 2)s-aCSF(?50ml /大鼠)经心脏灌注。
      注意:执行步骤A3的估计时间约为25秒。
    4. 立即取下头,迅速在中间做一个切口 的头皮,拉着皮肤,沿着颅骨切开 顶叶中线缝合。去除颅骨板, 小心地使用刮刀提取大脑,并将其转移到 氧化和预冷却的s-aCSF中约1分钟 ?分钟。
      注意:执行步骤A4的估计时间小于a 分钟。
    5. 将大脑放在冰上的玻璃板上(图1A)。 沿着纵向裂缝对脑进行半切(图1A,虚线 线1),并且相对于冠状面进行两个45度角切割 (图1A,虚线2和3),产生组织 块含有海马(图1B)。


      图1. 。 在一块玻璃板的Perfused老鼠脑子在冰。虚线表示 其中进行切割并且数字指示切割的顺序。乙。 阻塞的大脑与两个半球分离。 C.一个半球 大脑安装在安装盘上的Leica VT1200S振动 切片机
    6. 在中心放一小滴超级胶水 ?安装盘,小心地将一个半球放在胶水滴上 (图1C),并转移到切片阶段 ?振动切片机(图2A)

      图2。 A. Leica VT1200S 振动刀片切片机与阶段预冰与冰。乙。 使用切片机切割的两个横切片(300μm) 剩余的组织块。

    7. 立即用s-aCSF浆液覆盖组织并切割300μm切片(图2B)。
    8. 将部分转移到Netwell插入物的单独的孔中 (图3A),使用一次性转移移液管,并让部分 在含氧的常规aCSF中在34-35℃下回收30-60分钟。确保 所述部分平放在每个插入物的网格上,而不是 浮动或移动

      图3。 A.切片的恢复和治疗 建立。 B.在整合培养皿上显示单个切片 解剖海马。海马以虚线勾画 在切片的中间
    9. 恢复后,切片 准备实验治疗。治疗切片在氧合正常 aCSF与载体或感兴趣的药理学试剂。 治疗时间应根据经验确定(秒至分钟)。 每只动物的切片应均匀分布在载体和之间 ?治疗组。我们为每个样品集中四个切片,通常 每个样品产生300-400μg总蛋白。
    10. 治疗后, 使用一次性转移移液管,立即将切片转移入 整合培养皿中加入?20ml的0.2N高氯酸溶液 在常规aCSF中由70%高氯酸制备以稳定IP 3和 在室温下孵育2分钟。快速更换高氯酸-aCSF 与常规aCSF和将整体培养皿放在冰上。
    11. 从冰上的常规aCSF中的每个切片快速解剖海马 (图3B),使用具有弯曲针的1ml注射器, 针尖,迅速转移每个海马1.5毫升 微量离心管用300μl冰冷HEPES匀浆缓冲液 (每25ml缓冲液1片)。

  2. 组织匀浆和蛋白质定量
    1. 使用超声波仪在冰上匀浆组织(2次,3秒脉冲, 使用40%振幅设置),保持微量离心管 稳定,并且超声波探测器尖端以一半的方式浸入液体中 ?以避免起泡。
    2. 离心组织匀浆[3,000 rpm(1,000×g/g)在4℃下10分钟]以除去未破碎的细胞和细胞核。 ?丢弃沉淀。
    3. 测量每种蛋白质的浓度 样品。简言之,制备具有已知BSA的标准品 ?(1mg/ml原液)浓度和未知样品在双蒸 水。加入Bradford蛋白测定试剂,并在室温下孵育5 ?min。使用分光光度计测量595nm的吸光度。构建 标准曲线,使用来自标准品的吸光度值并确定 基于标准曲线的每个样品的蛋白质浓度。
    4. 将每个样品中的蛋白质浓度调整到相同的值(例如 2 ?μg/μl)使用具有蛋白酶的冰冷HEPES匀浆缓冲液 抑制剂。

  3. 测量IP 3 级别
    1. 用70%乙醇喷洒在台面上,彻底清洁。 确保在无尘环境中进行IP 3 测定
    2. 完全解冻IP <3>试剂盒试剂,并在使用前平衡至室温。
    3. 稀释IP sub 3标准品(20μM储备液)以制备10个3倍系列 使用IP <3>标准稀释缓冲液稀释。对于空白,请使用 ?IP 3 标准稀释缓冲液。
    4. 吸取10μl的空白, 标准品,每份样品(每份样品20μg蛋白质)一式三份 ?孔的384孔测定板具有非结合表面 阻挡尖端
    5. 向每个孔中加入10μlIP sub 3示踪剂,随后加入20μL μlIP 3 Sub结合蛋白,并通过上下吸移数次来充分混合 次。
    6. 仔细检查板的气泡。如果气泡形成, 他们用屏障移液器吸头。使用时避免交叉污染 每个井的新提示。将板在室温下孵育30分钟 ?在温和混合(?60rpm)的旋转器上黑暗
    7. 阅读 具有多模的IP 3+示踪剂的荧光偏振信号 酶标仪和荧光偏振滤光片,使用485nm 激发和530nm发射波长
    8. 绘制标准曲线 ?并计算每个样品中的IP 3 3浓度(nM范围内 海马)。除了绝对浓度值,IP <3>水平 对于每个车辆和实验组也可以报告相对 ?基线(无治疗对照)。

食谱

  1. 人工脑脊液(aCSF,应清新)
    126 mM NaCl 26mM NaHCO 3/v/v 10mM葡萄糖 3 mM KCl
    1.25mM NaH 2 PO 4>/
    2mM CaCl 2 2 / 1mM MgCl 2
    使用2N HCl或5N NaOH将pH调节至7.4 测量摩尔渗透压浓度(300-310 mOsm)
  2. 蔗糖aCSF(s-aCSF,应清新)
    75mM蔗糖 75mM NaCl 25mM NaHCO 3/v/v 15mM葡萄糖 2.4mM丙酮酸钠 2 mM KCl
    1.3mM抗坏血酸
    1.25mM NaH 2 PO 4>/
    3mM MgCl 2/
    0.5mM CaCl 2·h/v 使用2N HCl或5N NaOH将pH调节至7.4
  3. HEPES匀浆缓冲液(可在4℃储存至三个月)
    4mM HEPES(pH7.4) 320mM蔗糖 2 mM EGTA
    1mM原钒酸钠 50mM NaF 10mM焦磷酸钠 20 mM甘油磷酸盐 0.1mM PMSF
    在15 ml锥形管中使用前加入蛋白酶抑制剂混合物

致谢

这项研究得到国家卫生研究院资助R01 NS037324,妇女健康研究办公室和西北大学高通量分析设施的支持。

参考文献

  1. Tabatadze,N.,Huang,G.,May,R. M.,Jain,A.and Woolley,C.S。(2015)。 海马中抑制性突触的分子信号传导中的性别差异 J Neurosci 35(32):11252-11265。
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How to cite this protocol: Tabatadze, N. and Woolley, C. (2016). Measurement of Inositol Triphosphate Levels from Rat Hippocampal Slices. Bio-protocol 6(7): e1780. DOI: 10.21769/BioProtoc.1780; Full Text



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