بعد تضمين التعريف Immunogold من البروتينات متشابك في الثقافات شريحة الحصين
Summary
توطين وتوزيع البروتينات توفير معلومات هامة لفهم وظائفها الخلوية. يمكن استخدام دقة فائقة المكاني للالمجهر الإلكتروني (EM) لتحديد توطين التحت خلوية من مستضد معين بعد المناعية. لأنسجة الجهاز العصبي المركزي (CNS)، والحفاظ على السلامة الهيكلية بينما استضداد كان من الصعب الحفاظ على وخاصة في الدراسات EM. هنا، ونحن اعتماد إجراءات التي استخدمت للحفاظ على الهياكل ومولدات المضادات في الجهاز العصبي المركزي لدراسة وتوصيف البروتينات متشابك في قرن آمون الفئران الخلايا العصبية الهرمية CA1.
Abstract
Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.
Protocol
Representative Results
Discussion
في هذا البروتوكول، فقد اعتمدنا أسلوب Phend واينبرغ للدماغ والحبل الشوكي الأنسجة لدراسة العمود الفقري شجيري في قرن آمون شريحة الثقافات الفئران. العمود الفقري شجيري في منطقة قرن آمون CA3-CA1 هي الهياكل الحساسة التي تحتوي على مجموعة واسعة من البروتينات التي تلعب دورا هاما ف…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
فإن الكتاب أود أن أشكر فلورنسا ماثيو لإعداد الثقافات شريحة الحصين. وأيد هذا العمل من المنح المقدمة من المعهد الوطني الأمريكي للشيخوخة وجمعية الزهايمر لNZG.
Materials
| NAME OF REAGENT | COMPANY | CATALOG NUMBER | |
| 60 x 15 mm polystyrene Petri dish | Falcon | 351007 | |
| Disposable scalpel | EXELINT | 29552 | |
| Cell culture inserts | Millipore | PICM03050 | |
| 10 nm Goat-anti-rabbit gold | Electron Microscopy Sciences | 25108 | |
| Anti-Neurogranin antibody | Millipore | AB5620 | |
| 100% Picric acid | Electron Microscopy Sciences | 19550 | |
| 96% Paraformaldehyde | Acros Organics | AC41678-0030 | |
| 25% Glutaraldehyde (EM grade) | Sigma | G5882 | |
| Uranyl acetate | Electron Microscopy Sciences | 22400 | |
| p-Phenylenediamine | Sigma | P6001 | |
| Platinum (IV) chloride | Sigma | 379840 | |
| Tannic acid | Electron Microscopy Sciences | 21710 |
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