使用细胞色素线粒体呼吸功能的可视化 C氧化酶/琥珀酸脱氢酶（COX / SDH），双标组织化学
Summary
（COX / SDH），细胞色素C氧化酶/钠脱氢酶双标记方法允许直接可视化的新鲜冷冻组织切片中线粒体呼吸酶的缺陷。这是一个简单的组织化学技术，并在调查线粒体疾病，老化和衰老有关的疾病非常有用。
Abstract
线粒体DNA（mtDNA）的缺陷是疾病的重要原因，可能背后的老龄化和老龄化有关的改建1,2。衰老的线粒体理论表明mtDNA突变的作用，它可以改变生物能动态平衡和细胞的功能，在衰老过程中 3 。财富已编译的证据支持这一理论的1,4，例如线粒体DNA的mutator鼠标5;然而，线粒体老化损坏的确切作用是不完全了解6,7。
观察呼吸酶的活性，是一个简单的方法进行调查线粒体功能障碍。复杂的四，或细胞色素 c氧化酶（COX），是对线粒体功能至关重要。环氧合酶的催化亚基由线粒体DNA编码和复杂的装配（图1）是必不可少的。因此，适当的合成和功能主要是基于对线粒体DNA的完整性 2 。虽然可以调查其他呼吸道疾病复合物，复合物IV和II是最适合组织化学检查 8,9 。复杂的二，或琥珀酸脱氢酶（SDH），完全是由核DNA（图1）编码，其活动通常不会受损线粒体的影响，虽然增加可能表明线粒体生物合成10-12。观察受损的线粒体DNA在线粒体疾病，老化，与年龄有关的疾病往往导致低或缺乏COX活性2,12-14细胞的存在。虽然COX和SDH活动可以单独进行调查，15,16连续的双标记方法已被证明是在寻找与细胞线粒体功能障碍 12,17-21有利。
许多检测的最佳宪法已经确定，如底物浓度，电子受体/捐助者，中间电子载体，pH值的影响，反应吨IME 9,22,23。 3,3' -二氨基联苯胺（DAB）是一种有效和可靠的电子供体 22 。在运作的COX细胞，棕色indamine聚合物产品本地化线粒体嵴和饱和细胞22。因此，这些细胞功能失调的COX将不饱和民建联产品，硝基四氮唑（NBT），电子受体的减少SDH活性的可视化，一个蓝色的甲臜最终产品9,24。添加到细胞色素 C和琥珀酸钠基板内源性水平之间的控制和患病/突变体的组织 9正常化。过氧化氢 酶增加一条，作为一项预防措施，以避免可能的污染从过氧化物酶的活性 9,22反应。 methosulfate吩嗪（PMS），中间电子载体，是叠氮化钠，呼吸链抑制剂，结合使用，以增加形成的最终反应产物 9,25 。尽管这一通知ATION，影响这个看似简单的检测结果的一些关键细节，除了特异性的控制和在技术的进步，都尚未提出。
Protocol
1。组织编制为cryosectioning 按照与现有的伦理许可证牺牲的动物，无论是颈椎脱位或斩首。 快速收集利益的组织（如大脑），并迅速冻结干冰（组织可能需要用液态氮冷冻，以获得最佳的形态在戊烷或丙烷冻结）。铝箔存储于-80℃直至组织准备节。 cryosectioning冰冻组织嵌入在准备。 收集14微米的低温恒温器部分在-21 ° C（可能需要调整温度± 1-2 ° C）。解冻部分?…
Discussion
合并COX / SDH组织化学方法使细胞线粒体功能障碍的可视化。这种技术，与早期的研究可追溯到到1968年，仍然深受市民欢迎，许多考虑它的“金标准”，确定线粒体疾病患者14,19,26,27。现在常用的调查mtDNA突变驱动的老化和衰老相关的疾病 12,13,18,20,21,24 。 COX / SDH双标记方法经常被用来与其他技术的同时，确定具体的mtDNA突变，并进一步调查，如oximetric测量和分光光度酶分析2…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家衰老研究所（AG04418），国家药物滥用研究所，国立卫生卡罗林斯卡医学院研究生院合作计划研究院，卡罗林斯卡医学院，瑞典研究理事会，瑞典脑动力，和瑞典的脑基金会。非常感谢马蒂亚斯Karlen博士和朱Coppotelli与图1和2，分别创造性的支持;卡琳Pernold技术援助和Drs。巴里J. Hoffer，拉尔斯奥尔森和尼尔斯戈兰拉尔森许多有益的建议和讨论。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Dry Ice | AGA Gas AB | block form | |
| Isopentane (2-methylbutane) | Sigma-Aldrich | 277258 CAS: 78-78-4 |
|
| Cyrostat embedding solution | Sakura Finetek | Tissue Tek 4583 | |
| Cryostat | Microm | Microm Model HM 500M | |
| Slides | Thermo Scientific | Super Frost Plus Menzel Gläser J1800AMWZ |
|
| Cover glasses Borosilicate glass |
VWR International | 16004-098 | 24 x 50 mm |
| Filter Paper | Munktell Filter AB | Quality: 1350 Article Number: 242 001 |
430 x 430 mm |
| 3,3′-diaminobenzidine tetrahydrochloride (DAB) | Sigma-Aldrich | Sigma Liquid Substrate System, D7304 | |
| Cytochrome c (Type III, from equine heart) | Sigma-Aldrich | C2506 CAS: 9007-43-6 |
|
| Bovine catalase (from liver) | Sigma-Aldrich | C9322 CAS: 9001-05-2 |
|
| Nitroblue tetrazolium (NBT) | Sigma-Aldrich | N6876 CAS: 298-83-9 |
|
| Sodium succinate | Sigma-Aldrich | S2378 CAS: 6106-21-4 |
|
| Phenazine methosulfate (PMS) | Sigma-Aldrich | P9625 CAS: 299-11-6 |
PMS is light sensitive. Shield from light. |
| Sodium azide | Sigma-Aldrich | S8032 CAS: 26628-22-8 |
|
| Xylene | VWR International | EM-XX0060-4 | |
| Entellan | VWR International | 100503-870 | |
| Malonate (Malonic acid) |
Sigma-Aldrich | M1296 CAS: 141-82-2 |
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Tags
Mitochondrial Respiratory FunctionCytochrome C OxidaseSuccinate DehydrogenaseCOX/SDH Double-labeling HistochemistryMitochondrial DNA DefectsMtDNA MutationsBioenergetics HomeostasisCellular FunctionMitochondrial Theory Of AgingMtDNA DamageRespiratory EnzymesCytochrome C Oxidase (COX)MtDNA IntegritySuccinate Dehydrogenase (SDH)Nuclear DNAMitochondrial DiseasesAgingAge-related Diseases
Cite This Article
Ross, J. M. Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry. J. Vis. Exp. (57), e3266, doi:10.3791/3266 (2011).