ELISA 组织样本的处理—大鼠组织

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大鼠肝脏匀浆

方法一

  • 提取缓冲液:PBS,pH 7.2,0.05%叠氮钠、0.5%Triton X-100,和蛋白酶抑制剂混合物
  • 步骤:脂多糖(10 mg/mL生理盐水),按15毫克/千克静脉注射以处死老鼠。杀死后,迅速切除肝脏,冲洗血液,在预冷提取缓冲液中研磨。然后超声处理10分钟。匀浆在12000 x g下离心10分钟。取上清备用。
  • 参考文献:
    Reference: Borovikova, L., S. Ivanova, M. Zhang, H. Yang, G.I. Botchkina, L.R. Watkins, H. Wang, N. Abumrad, J.W. Eaton, and K.J. Tracey (2000) Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature 405: 458-462 (cites the use of KRC3012 with liver tissue homogenates).

方法二

  • 提取缓冲液:磷酸盐缓冲盐,13μL/mL蛋白酶抑制剂,5%胎牛血清
  • 步骤:在干冰上将肝脏切成约500毫克的切片,然后放入2mL冰浴预冷磷酸盐缓冲盐中冰浴研磨,然后用相同的溶液稀释至5 mL。然后1500 x g,离心15分钟。取澄清的上清液分为1ml的小份,并在-70℃下储存备用。
  • 参考文献:
    Lennie, T.A., M.D. Mortman, and R.J. Seeley (2001) Activity of body energy regulatory pathways in inflammation-induced anorexia. Physiology and Behavior 73(4):517-523 (cites the use of KRC0062 with serum and with tissue homogenates).

方法三

  • 提取缓冲液:0.242% Tris,5 mg/mL aprotinin, 5 mg/mL leupeptin, 1 mg/mL pepstatin, 10 mg/mL phenylmethylsulfonylfluoride(溶于异丙醇)
  • 步骤:将肝脏或肺样本(0.1 g)加1 mL冰浴提取缓冲液,冰浴研磨。2100 rpm,离心20分钟。上清冻存备用。
  • 参考文献:
    Jho, D.H., T.A. Babcock, R. Tevar, W.S. Helton, and H.J. Espat (2002) Eicosapentaenoic acid supplementation reduces tumor volume and attenuates cachexia in a rat model of progressive non-metastasizing malignancy. Journal of Parenteral and Enteral Nutrition 26(5):291-297

大鼠脾匀浆术

  • 提取缓冲液:磷酸盐缓冲液(pH7.2),0.05%叠氮化钠,0.5%Triton X 100,1 mM PMSF,1 mg/mL胃蛋白酶抑制剂A(pepstatin A)、1μg/mL 抑肽酶(aprotinin)和1μg/mL亮肽(leupeptin)
  • 步骤:对冷冻组织样本进行称重,并按100 mg组织加1 mL的量加提取缓冲液。研磨,并进行一轮冻融,然后超声处理10分钟,4℃下孵育1小时。所得匀浆120000 x g下离心。上清液-80°C储存备用。
  • 参考文献:
    Molina, P.E. (2001) Opiate modulation of hemodynamic, hormonal, and cytokine responses to hemorrhage. Shock 15(6): 471-478.
    Molina, P.E. (2001) Noradrenergic inhibition of TNF upregulation in hemorrhagic shock. Neuroimmunomodulation 9(3):125-133.

大鼠肺组织制备法

方法一

  • 提取缓冲液:10 mM HEPES、10 mM KCl、0.5 M sucrose、1 mM EGTA、1 mM DTT。
  • 步骤:将肺组织于5体积的缓冲液中研磨匀浆。匀浆750 x g离心10分钟以分离细胞核。含有细胞质成分的上清,-80°C储存备用。
  • 参考文献:
    Reference: Calkins, C.M., D.D. Bensard, J.K. Heimbach, X. Meng, B.D. Shames, E.J. Pulido, R.C. McIntyre (2001) L-arginine attenuates lipopolysaccharide induced lung chemokine production. Am. J. Physiol. Lung Cell Mol. Physiol. 280(3):L400-408 (uses KRC1022 rat MIP-2 ELISA kit).

方法二

  • 提取缓冲液:0.5%Triton X-100、150 mM NaCl、15 mM Tris、1 mM CaCl和1 mM MgCl2(pH 7.4)
  • 步骤:收集支气管肺泡灌洗液后,取左肺,加1 mL提取缓冲液,使用组织匀浆器(Biospec产品,Racine,WI)进行研磨。然后10000 x g离心10分钟。上清液-80°C储存备用。
  • 参考文献:
    Reference: Whitehead, G.S.; K.A. Grasman and E.C. Kimmel (2003) Lung function and airway inflammation in rats following exposure to combustion products of carbon-graphite/epoxy composite material: comparison to a rodent model of acute lung injury. Toxicology 183(1-3):175-197 (cites the use KRC1022 with BALF and lung tissue homogenates).

方法三

  • 提取缓冲液:磷酸盐缓冲液(pH7.2),0.05%叠氮化钠,0.5% Triton X-100,及蛋白酶抑制剂组合:1 mM 苯甲基磺酰氟, 1 μg/mL peptstain A, 1 μg/mL aproptinin及1 μg/mL leupeptin)
  • 步骤:取冻存大鼠肺组织,按每100 mg加1 mL 4°C预冷的提取缓冲液,研磨匀浆,并采取一轮冻融,超声处理10分钟,在4°C下孵育1小时的处理。最后的匀浆 120000 x g(超速离心)离心。组织上清液用于细胞因子测定。
  • 参考文献:
    Reference: Phelan, H., P. Stahls, J. Hunt, G.J. Bagby, and P.E. Molina (2002) Impact of alcohol intoxication on hemodynamic, metabolic, and cytokine responses to hemorrhagic shock. J. Trauma- Injury Infection and Critical Care 52(4):675-682 (cites the use of KRC3012, KRC0812, KRC0062, and KRC0102) with lung tissue homogenates and plasma samples).

方法四

  • 提取缓冲液:10 mM HEPES(N-2-hydroxyethylpiperazine-N-2- ethanesulfonic acid,pH 7.9)、10 mM KCl、0.1 mM EGTA(ethyleneglycol-bis-(beta-aminoethylether)- n,n’-tetraacetic acid),1 mM DTT(dithiothreitol),0.5 mM苯甲基磺酰氟(phenylmethylsulfonyl fluoride)和0.6% N-40替代物(octylphenoxy-polyethoxy-ethanol,Nonidet P-40)
  • 步骤:将来自下三分之一和上三分之一肺的冰冻组织在提取缓冲液中超声匀浆。4℃下12000 rpm离心10分钟。上清液-80°C储存备用。
  • 参考文献:
    Perrot, M., Y. Imai, G.A. Volgyesi, T.K. Waddell, M.Y. Liu, J.B. Mullen, K. McRae, H.B. Zhang, A.S. Slutsky, V.M. Ranieri and S. Keshavjee (2002) Effect of ventilator-induced lung injury on the development of reperfusion injury in a rat lung transplant. Journal of Thoracic and Cardiovascular Surgery 124(6):1137-1144

方法五

  • 提取缓冲液:0.242% Tris,5 mg/mL aprotinin, 5 mg/mL leupeptin, 1 mg/mL pepstatin, 10 mg/mL phenylmethylsulfonylfluoride(溶于异丙醇)
  • 步骤:将肝脏或肺样本(0.1 g)加1 mL冰浴提取缓冲液,冰浴研磨。2100 rpm,离心20分钟。上清冻存备用。
  • 参考文献:
    Jho, D.H., T.A. Babcock, R. Tevar, W.S. Helton, and H.J. Espat (2002) Eicosapentaenoic acid supplementation reduces tumor volume and attenuates cachexia in a rat model of progressive non-metastasizing malignancy. Journal of Parenteral and Enteral Nutrition 26(5):291-297.

方法六

  • 提取缓冲液:磷酸盐缓冲液(pH7.2),0.05%叠氮化钠,0.5%Triton X 100,1 mM PMSF,1 mg/mL胃蛋白酶抑制剂A(pepstatin A)、1μg/mL 抑肽酶(aprotinin)和1μg/mL亮肽(leupeptin)
  • 步骤:对冷冻组织样本进行称重,并按100 mg组织加1 mL的量加提取缓冲液。研磨,并进行一轮冻融,然后超声处理10分钟,4℃下孵育1小时。所得匀浆120000 x g下离心。上清液-80°C储存备用。
  • 参考文献:
    Ø Molina, P.E. (2001) Opiate modulation of hemodynamic, hormonal, and cytokine responses to hemorrhage. Shock 15(6): 471-478.
    Ø Molina, P.E. (2001) Noradrenergic inhibition of TNF upregulation in hemorrhagic shock. Neuroimmunomodulation 9(3):125-133.

大鼠脑匀浆程序

方法一

  • 提取缓冲液:HEPES 25 mM, pH 7.4, 3-[(3-cholamidopropyl) dimethyl-ammonio]1-propanesulfonate 0.1%, MgCl2 5 mM, EDTA 1.3 mM, EGTA 1 mM, 10 μg/mL pepstatin, aprotinin, and leupeptin, and 1 mM PMSF。
  • 步骤:分别解剖大脑半球皮质,并于冰浴预冷提取缓冲液中研磨匀浆。匀浆50000 rpm,离心15分钟。取上清-80°C下储存备用。用于测定IL-1β、IL-10、MCP-1、MIP-2和TNF-α的含量。
  • 参考文献:
    Rabuffetti, M., C. Sciorati, G. Tarozzo, E. Clementi, A.A. Manfredi, and M. Beltramo (2000) Inhibition of caspase-1-like activity by Ac-Tyr-Val-Ala-Asp-chloromethyl ketone induces long-lasting neuroprotection in cerebral ischemia through apoptosis reduction and decrease of proinflammatory cytokines. J. Neurosci. 20(12):4398-4404

方法二

  • 提取缓冲液:0.1 M冰磷酸盐缓冲液(pH值7.4)。
  • 步骤:用冰镇等渗盐水(0.9% w/v NaCl)冲洗离体脑。解剖大脑海马区,并使用10倍(w/v)体积0.1 M冰浴预冷磷酸盐缓冲液(pH值7.4)均质化,然后在10000 g下离心15分钟(4°C)。上清分装备用。利用ELISA试剂盒检测GCSF和 GFAP的含量。
  • 参考文献:
    Sharma, N. and A. B. Gaikwad (2020). "Effects of renal ischemia injury on brain in diabetic and non-diabetic rats: Role of angiotensin II type 2 receptor and angiotensin-converting enzyme 2." Eur J Pharmacol 882: 173241.

大鼠肠粘膜匀浆的制备方法

  • 提取缓冲液:Tris-EDTA (10 mM Tris-HCl, 1 mM EDTA, pH 7.4)溶液中含有0.05%叠氮化钠,1%吐温-80,2 mM PMSF,1ug/mL 以下蛋白酶抑制剂:aprotinin, leupeptin, pepstatin A
  • 步骤:用玻片刮去下层肌肉层的粘膜。粘膜细胞加提取缓冲液。研磨20秒。离心匀浆(11000 x g,4°C,10分钟)。收集上清液并用4.5微米过滤器过滤上清液。过滤所得上清液备用。
  • 参考文献:
    Ø Castagliuolo, I., A.C. Keates, C.C. Wang, A, Pasha, L. Valenick, C.P. Kelly, S.T. Nikulasson, J.T. LaMont, and C. Pothoulakis (1998) Clostridium difficile toxin A stimulates macrophage-inflammatory protein-2 production in rat intestinal epithelial cells. J. Immunol. 160:6039-6045.
    Ø Castagliulo, I., K. Karalis, L. Valenick, A. Pasha, S. Nikulasson, M. Wlk, and C. Pothoulakis (2001) Endogenous corticosteroids modulate Clostridium difficile toxin A-induced enteritis in rats. Am. J. Physiol. Gastrointest. Liver Physiol. 280:G539-545.

大鼠脊髓匀浆术

方法一

  • 提取缓冲液:细胞培养基(RPMI+10%热灭活胎牛血清)
  • 步骤:称重脊髓样本,然后在-70°C下冷冻。每克脊髓加冰浴预冷9 mL细胞培养基,并于冰浴下超声匀浆。匀浆在4°C,1310 x g,离心15分钟。立即使用上清液进行IL-1β测定。
  • 参考文献:
    Wang, C.X., J.A. Olschowka, and J.R. Wrathall (1997) Increase of IL-1beta mRNA and protein in the spinal cord following experimental traumatic injury in the rat. Brain Research 759:190-196.

方法二

  • 提取缓冲液:50 mM NaCl,10 mM Tris,2.5 mM MgCl2,pH 7.4,蛋白酶抑制剂(德国Boehringer Mannheim)1片/50 ml
  • 步骤:将一根18号针插入脊柱的尾端并用冰冷的PBS分离取出脊髓。脊髓在干冰或者液氮中速冻,-80℃下保存备用。从完整的冷冻脊髓中取出0.5厘米的腰椎脊髓段(包括L4/L5节段)并称重。将组织切碎并放入0.25 ml提取缓冲液。研磨均匀。匀浆在4℃下,20,000 g,离心30分钟。等分上清液并储存在-80℃下用于细胞因子的检测。
  • 参考文献:
    Ø Sweitser, S., D. Martin, J. DeLeo (2001) Intrathecal interleukin-1 receptor antagonist in combination with soluble tumor necrosis factor receptor exhibit an anti-alppdynic action in a rat model of neurophathic pain. Neuroscience 103:529-539.
    Ø Sweitzer, S.M. and J.A. DeLeo (2002) The active metabolite of leflunomide, an immunosuppressive agent, reduces mechanical sensitivity in a rat mononeuropathy model. Journal of Pain 3(5):360-368.

方法三

  • 提取缓冲液:磷酸盐缓冲盐水
  • 步骤:新鲜分离的脊髓在1 ml无菌磷酸盐缓冲盐水中超声破碎。400 x g下离心10分钟。上清液立即在-80°C下冷冻备用。
  • 参考文献:
    Ø References: Adamus, G., M. Manczak, and M. Machnicki (2001) Expression of CC chemokines and their receptors in the eye in autoimmune anterior uveitis associated with EAE. Investigative Opthalmology and Visual Science 42(12):2894-2903.
    Ø Manczak, M., S.G. Jiang, B. Orzechowska, and G. Adamus (2002) Crucial role of CCL3/MIP-1 alpha in the recurrence of autoimmune anterior uveitis induced with myelin basic protein in Lewis rats. J. Autoimmunity 18(4):259-270.

大鼠皮肤组织处理方法

方法一

  • 提取缓冲液:10 mm Tris pH 7.4,150 mm NaCl,1%Triton X-100
  • 步骤:用打孔器取6 mm皮肤。按每克组织加1.5 mL提取缓冲液,然后研磨为匀浆。将匀浆转移到1.5 mL 离心管中,在4°C下13000 x g离心10分钟。上清液-80°C储存备用。
  • 参考文献:
    Reference: Blalock, T.D., J.C. Varela, S. Gowda, Y. Tang, C. Chen, B.A. Mast, and G.S. Schultz (2001) Ischemic skin wound healing models in rats. Wounds 13(1):35-44.

方法二

  • 提取缓冲液:PBS加蛋白酶抑制剂
  • 步骤:移植后的不同时间点,取皮瓣组织,立即液氮冻存。检测当天,让活组织解冻,取20-50 mg,加提取液研磨。匀浆10000 x g离心20分钟以去除碎屑和不溶性物质。上清液一部分用于BCA法测总蛋白含量,一部分用ELISA法测定大鼠IFN-γ。
  • 参考文献:Fernandez-Botran, R., V. Gorantla, X.C. Sun, X.P. Ren, G. Perez-Abadia, F.A. Crespo, R. Oliver, H.I. Orhu, E.E. Quan, C. Maldonado, M. Ray, and J.H. Barker (2002) Targeting of glycosaminoglycan-cytokine interactions as a novel therapeutic approach in allotransplantation. Transplantation 74(5):623-629 (cites the use of KRC4022 with skin biopsies).