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.

1. تثبيت مثبتات مسببة للسرطان؛ ارتداء القفازات والتعامل مع مثبتات في غطاء الدخان. ما لم يذكر خلاف ذلك، وتتم جميع حضانات على الجليد، وينبغي أن تتم تصفيته قبل الاستعمال كل الحلول. استخدام الإلكترون المجهري الصف الكواشف. يوم 1 <ol style=";text-align:right;direction:rtl"><li class="jove_content" style=";text-align:right;direction:rtl"> بعد الظروف التجريبية (حقن الفيروسية مثل العلاج من تعاطي المخدرات)، ضع الغشاء مع شرائح قرن آمون في عضوي النمط لمدة 60 × 15 مم طبق بتري تحتوي على الجليد البوليسترين الباردة الفوسفات 0.1 M عازلة (عازلة للفوسفات سورنسن)، ودرجة الحموضة 7.3. إضافة 1-2 مل من 0.1 العازلة الفوسفات M مباشرة على أعلى من شرائح للحفاظ على الباردة. </li></ol> خطوة حاسمة: استخدم دوما طازجة أعدت مخازن ومثبتات. تأكد من أن الرقم الهيدروجيني للالعازلة هو داخل النطاق المطلوب. ويمكن لعدم القيام بذلك تلف الهياكل الخلوية. <ol start="2" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لعزل الحقل الفرعي CA1 للشريحة، استخدممشرط لقطع القابل للتصرف بلطف عبر شريحة بجوار DG، بالتوازي مع طبقة الخلايا CA1 (الشكل 1). ثم قص شريحة المتبقية عموديا لإزالة الحقل الفرعي CA3 ومرفد و. قطع زاوية من شريحة للمساعدة في تحديد السطح العلوي للنسيج. </li></ol> ملاحظة: في حالة تسليم أي مطلوب من البروتين الفيروسي من الفائدة، يمكن ان تكون ثابتة في شريحة الأنسجة كامل بعد إزالة وسائل الإعلام مع طيف شطف من الجليد الباردة العازلة. ثم اعقب ذلك عن طريق الاستغناء عن CA1. خطوة حاسمة: تتبع الجانب العلوي من الأنسجة من أجل باجتزاء السليم للشبكات في وقت لاحق. <ol start="3" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة الأنسجة بعناية من الغشاء باستخدام مساعدات من مشرط. استخدام ماصة باستور لنقل بلطف الأنسجة إلى لوحة تحتوي على 12-جيدا العازلة الفوسفات 0،1 M. </li><li style=";text-align:right;direction:rtl"> إزالة المخزن المؤقت في البئر من لوحة وإضافة 500 &amp; Mش؛ L - 1 مل من الجليد الباردة مثبت (الرقم الهيدروجيني 7.3) تتألف مما يلي: 0.1٪ حمض البكريك، بارافورمالدهيد 1٪، وغلوتارالدهيد 2.5٪ في 0.1 العازلة الفوسفات M. احتضان لمدة 2 ساعة في 4 درجات مئوية. </li></ol> ملاحظة: قد يكون حمض البكريك متفجرة عندما تجف. يبقيه مبلل بالماء في حاوية مغلقة بإحكام. تخزينها في مكان جاف وجيد التهوية. <ol start="5" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة مثبت من البئر وغسل العينات 3 مرات (20 دقيقة لكل منهما) في 0.1 العازلة الفوسفات M. </li><li style=";text-align:right;direction:rtl"> احتضان لمدة 40 دقيقة في حمض التانيك 1٪ (W / V) في 0.1 العازلة ماليات M، 6.0 درجة الحموضة. </li><li style=";text-align:right;direction:rtl"> شطف مرتين (20 دقيقة لكل منهما) في المخزن المؤقت ماليات. </li><li style=";text-align:right;direction:rtl"> احتضان لمدة 40 دقيقة في خلات اليورانيل 1٪ (W / V) في المخزن المؤقت ماليات في الظلام. خلات اليورانيل حساسة للضوء وغير المشعة. تغطية الكأس مع Parafilm عندما حل وتخزين أي العازلة المستخدمة في الظلام في C. ° 4 </li><li style=";text-align:right;direction:rtl"> شطف مرتين (20 دقيقة لكل منهما) في المخزن المؤقت ماليات. </li><li style=";text-align:right;direction:rtl"> احتضان لمدة 20 دقيقة في الكلور البلاتين 0.5٪IDE (ث / ت) في المخزن المؤقت ماليات. </li><li style=";text-align:right;direction:rtl"> شطف مرتين (20 دقيقة لكل منهما) في المخزن المؤقت ماليات. تخزين العينات في 4 درجات مئوية حتى تكون جاهزة لمزيد من المعالجة. </li></ol> 2. الجفاف وتضمينها أكسيد البروبيلين هو مسرطنة. تجنب أبخرة من خلال العمل معها في غطاء الدخان. استخدام المطلقة، والإيثانول النقي 100٪ يحتوي على أي أثر للماء. هذا تمييع الإيثانول المطلق لإنتاج تركيزات مختلفة للجفاف. يوم 2 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> احتضان لمدة 5 دقائق في الايثانول 50٪ ثم لمدة 5 دقائق في الايثانول 70٪. </li><li style=";text-align:right;direction:rtl"> احتضان لمدة 15 دقيقة في٪ 1 الطازجة ف فينيلين (PPD) في الايثانول 70٪. </li><li style=";text-align:right;direction:rtl"> شطف ثلاث مرات في الايثانول 70٪. </li><li style=";text-align:right;direction:rtl"> احتضان لمدة 5 دقائق في الايثانول 80٪ ثم لمدة 5 دقائق في الايثانول 95٪. </li><li style=";text-align:right;direction:rtl"> احتضان في الإيثانول بنسبة 100٪ مرتين كل 5 دقائق. </li><li style=";text-align:right;direction:rtl"> إعداد قوارير الزجاج مع الأغطية الملولبة والتأكد من أنها نظيفة وجافة تماما. نقل قوارير الزجاج إلى شرائح وتسمية كل فيال ببساطة ووضوح. </li><li style=";text-align:right;direction:rtl"> احتضان لمدة 5 دقائق في 01:01 الإيثانول: أكسيد البروبيلين. </li><li style=";text-align:right;direction:rtl"> احتضان أكسيد البروبيلين في 100٪ مرتين كل 5 دقائق. </li><li style=";text-align:right;direction:rtl"> إضافة الراتنج (EPON) إلى كل قارورة لجعل مزيج 1:1 مع أكسيد البروبيلين. المزيج بلطف لمدة 2 ساعة. لا يهز قنينة بدقة جدا لمنع الفقاعات التي يمكن أن تتداخل مع التضمين. </li><li style=";text-align:right;direction:rtl"> إضافة الراتنج لجعل خليط مع أكسيد البروبيلين 03:01، ومزيج لمدة 2 ساعة. </li><li style=";text-align:right;direction:rtl"> نقل الراتنج إلى 100٪ واحتضان O / N. </li></ol> اليوم 3 <ol start="12" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ساندويتش شرائح عينات من البلاستيك بين ACLAR وعلاج ل24 ساعة عند 60 ° C. </li></ol> 3. باجتزاء وتركيب شبكات على يوم 4 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قطع شبه رقيقة (0.5 ميكرومتر) وصمة عار مع أقسام 1٪ + البوراكس زرقة الطولويدين 1٪ لتحديد الاتجاه الصحيح من العينات. </li><li style=";text-align:right;direction:rtl"> قطع المقاطع سامسونج (60 نانومتر) وجبل على شبكات النيكل، مقطع واحد في الشبكة. </li></ol>4. المناعية يجب أن تتم تصفيته جميع مخازن والماء قبل الاستخدام. اليوم 5 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع قطرة (~ 50 ميكرولتر) من العازلة Tween-20/phosphate 1٪ (T / PB)، ودرجة الحموضة 7.5، على قطعة من Parafilm نظيفة على سطح مستو. اختيار ما يصل بلطف الشبكة مع ملقط على حافة نظيفة وتطفو على المخزن المؤقت، القسم الأسفل، واحتضان لمدة 10 دقيقة في RT. </li><li style=";text-align:right;direction:rtl"> استنزاف السائل الزائد من الشبكة عن طريق وضع الجانب القسم حتى على ورقة لتصفية ثم تطفو على جليكاين 50 مم في T / PB لمدة 15 دقيقة في RT. </li></ol> خطوة حاسمة: لتجنب الإفراط في &#39;ترحيل&#39; من الحلول من حل قطرة واحدة إلى أخرى خلال حضانات، واستنزاف السائل الزائد عن طريق لمس برفق على حافة الشبكة على الورق 1 # التصفية بين كل تغيير الحل. أيضا إزالة أي حل محاصرين بين أحضان EM ملقط عقد الشبكة من فتل بلطف مع قطعة من ورق الترشيح بين الأسلحة ملقط بينما ل ensuدق أبواب لا تجف تماما. <ol start="3" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تجفيف الشبكة مع ورقة الترشيح ثم تطفو على منع محلول يحتوي على 2.5٪ BSA والمصل 2.5٪ من الحيوانات من الأجسام المضادة الثانوية في T / PB لمدة 30 دقيقة في RT. </li><li style=";text-align:right;direction:rtl"> احتضان مع الأجسام المضادة الأولية (في T / PB) في RT أو O / N ° 4 في C. الوقت الأمثل ودرجة حرارة الحضانة، فضلا عن تركيز الأجسام المضادة، يجب أن يتم تحديد تجريبيا. </li><li style=";text-align:right;direction:rtl"> غسل الشبكة ثلاث مرات (2 دقيقة لكل منهما) مع T / PB. </li><li style=";text-align:right;direction:rtl"> احتضان الضد الثانوية مع (1:20 المضادة للأرنب أو مكافحة الماوس بالإضافة إلى الذهب نانومتر 10) ل1 ساعة في RT. </li><li style=";text-align:right;direction:rtl"> يغسل ثلاث مرات (2 دقيقة لكل منهما) مع T / PB. </li><li style=";text-align:right;direction:rtl"> POSTFIX مع غلوتارالدهيد 2٪ في T / PB لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> يغسل ثلاث مرات (2 دقيقة لكل منهما) مع T / PB ثم ثلاث مرات (2 دقيقة لكل منهما) مع الماء المصفى. </li><li style=";text-align:right;direction:rtl"> احتضان مع خلات اليورانيل 2٪ في الماء لمدة 10 دقيقة. </li></ol> في حين أن الشبكة هو تلطيخ، وإعداد CO 2-مجانا من قبل غرفة placinGA قطعة من Parafilm في وسط طبق بتري الزجاج. ثم ضع 4-6 كريات هيدروكسيد الصوديوم في طبق في جميع أنحاء Parafilm لامتصاص CO 2 في الهواء. الحفاظ على أعلى إغلاق لبضع دقائق. استخدام ماصة باستور ونقل بسرعة حجم صغير من محلول سترات رينولد يؤدي إلى Parafilm في طبق بتري الزجاج. فتح الجزء العلوي يكفي لإدراج ماصة باستور للحد من إعادة إدخال CO 2 في الغرفة. ملاحظة: لجعل حل رينولد سترات الرصاص، يغلي 100 مل من الماء منزوع الأيونات في فرن ميكروويف والسماح لتبرد في وعاء محكم. في قارورة 50 مل الحجمي بالسدادة، مزيج الرصاص 1،33 ز نترات، 1،76 ز سترات الصوديوم و 30 مل من الماء المغلي عن طريق هز بقوة لمدة 1 دقيقة. ثم يهز بشكل متقطع لمدة 30 دقيقة. لهذا الحل غائم، إضافة 8 مل من 1 M هيدروكسيد الصوديوم وعكس ببطء القارورة عدة مرات. والحل أصبح واضحا. تقديم حل ل50 مل مع وا المغليثالثا. يخزن المحلول مغلقة بإحكام. إذا راسب يظهر، تجاهل وجعل واحدة جديدة. <ol start="11" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في ثلاثة أكواب أصغر إعداد الدافئة، والمياه المغلية الطازجة منزوع الأيونات. يغسل خلات اليورانيل بواسطة غمس الشبكة في الدورق الأول ودوامة برفق لمدة 30 ثانية حول. كرر هذه الخطوة في الأكواب الأخريين. </li><li style=";text-align:right;direction:rtl"> فتح الجزء العلوي من الزجاج طبق بتري يكفي لوضع الشبكة في قطرة من محلول سترات رينولد الرصاص. إذا كان ذلك ممكنا، وارتداء قناع أثناء القيام هذا لتجنب التنفس على الرصاص وسترات لمنع تشكيل راسب. احتضان لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> فتح الجزء العلوي يكفي لإزالة الشبكة. تجنب التنفس على سترات الرصاص. غسل الشبكة ثلاث مرات متتالية عن طريق غمس ذلك في ثلاثة أكواب الماء الدافئ مع، المقطر المغلي طازجة. السماح للشبكة الجافة على قطعة من ورق الترشيح، قسم أعلى. العينة هو الآن على استعداد للالمجهر الإلكتروني. </li></ol>

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R., Lonnie, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+2.">Ch. 2.</a> Electron Microscopy. 30, (1992).</li><li>Cho, K. O., Hunt, C. A., Kennedy, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+brain+postsynaptic+density+fraction+contains+a+homolog+of+the+Drosophila+discs-large+tumor+suppressor+protein.">The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.</a> Neuron. 9, 929-942 (1992).</li><li>Hunt, C. A., Schenker, L. J., Kennedy, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PSD-95+is+associated+with+the+postsynaptic+density+and+not+with+the+presynaptic+membrane+at+forebrain+synapses.">PSD-95 is associated with the postsynaptic density and not with the presynaptic membrane at forebrain synapses.</a> J. Neurosci. 16, 1380-1388 (1996).</li><li>Gispen, W. H., Leunissen, J. L., Oestreicher, A. B., Verkleij, A. J., Zwiers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+localization+of+B-50+phosphoprotein:+the+(ACTH)-sensitive+protein+kinase+substrate+involved+in+rat+brain+polyphosphoinositide+metabolism.">Presynaptic localization of B-50 phosphoprotein: the (ACTH)-sensitive protein kinase substrate involved in rat brain polyphosphoinositide metabolism.</a> Brain Research. 328, 381-385 (1985).</li><li>Biewenga, J. E., Schrama, L. H., Gispen, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+phosphoprotein+B-50/GAP-43+in+neuronal+and+synaptic+plasticity.">Presynaptic phosphoprotein B-50/GAP-43 in neuronal and synaptic plasticity.</a> Acta Biochim. Pol. 43, 327-338 (1996).</li><li>Maunsbach, A. A., Bjorn, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+16.">Ch. 16.</a> Biomedical Electron Microscopy Illustrated Methods and Interpretations. , 383-426 (1999).</li></ol>

ليس لدينا شيء في الكشف عنها.

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...

<table border="1">
 <tbody>
 <tr>
 <td>NAME OF REAGENT</td>
 <td>COMPANY</td>
 <td>CATALOG NUMBER</td>
 <td></td>
 </tr>
 <tr>
 <td>60 x 15 mm polystyrene Petri dish</td>
 <td>Falcon</td>
 <td>351007</td>
 <td></td>
 </tr>
 <tr>
 <td>Disposable scalpel</td>
 <td>EXELINT</td>
 <td>29552</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell culture inserts</td>
 <td>Millipore</td>
 <td>PICM03050</td>
 <td></td>
 </tr>
 <tr>
 <td>10 nm Goat-anti-rabbit gold</td>
 <td>Electron Microscopy Sciences</td>
 <td>25108</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Neurogranin antibody</td>
 <td>Millipore</td>
 <td>AB5620</td>
 <td></td>
 </tr>
 <tr>
 <td>100% Picric acid</td>
 <td>Electron Microscopy Sciences</td>
 <td>19550</td>
 <td></td>
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 <td>96% Paraformaldehyde</td>
 <td>Acros Organics</td>
 <td>AC41678-0030</td>
 <td></td>
 </tr>
 <tr>
 <td>25% Glutaraldehyde (EM grade)</td>
 <td>Sigma</td>
 <td>G5882</td>
 <td></td>
 </tr>
 <tr>
 <td>Uranyl acetate</td>
 <td>Electron Microscopy Sciences</td>
 <td>22400</td>
 <td></td>
 </tr>
 <tr>
 <td>p-Phenylenediamine</td>
 <td>Sigma</td>
 <td>P6001</td>
 <td></td>
 </tr>
 <tr>
 <td>Platinum (IV) chloride</td>
 <td>Sigma</td>
 <td>379840</td>
 <td></td>
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 <td>Tannic acid</td>
 <td>Electron Microscopy Sciences</td>
 <td>21710</td>
 <td></td>
 </tr>
 </tbody>
</table>

post-embedding immunogold labeling of synaptic proteins in hippocampal slice cultures

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.

الرقم 2B مثالا على توزيع جزيئات نج المحلية في العمود الفقري شجيري الخلايا العصبية الهرمية CA1 قرن آمون. تمت تغطية شبكات النيكل مع الأنسجة (60 نانومتر) التي تحتوي على سامسونج CA1 منطقة قرن آمون (كما رأينا في الشكل 2A) في 1٪ T / PB، 50 مم جليكاين، ومنعت بعد ذلك مع ...

Watch this Scientific Journal Video about Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures at JoVE.com

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

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.

بعد تضمين التعريف Immunogold من البروتينات متشابك في الثقافات شريحة الحصين

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

post-embedding-immunogold-labeling-synaptic-proteins-hippocampal

Research

JoVE Journal

Neuroscience

17.0K Views.  Medical College of Wisconsin. 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 الص...

Video: Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

إعداد Oligomeric β اميلويد 1-42 والتعريفي للعاهة اللدونة متشابك على شرائح الحصين

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

وعضوي النمط الثقافة شريحة الحصين النموذجي لفحص الإصابات متعلق بالخلايا العصبية

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

عضوي النمط الثقافات شريحة الحصين

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

على تحليل من الخليوي بركينيي علم الصرف شجيرى في الثقافات شريحة عضوي النمط

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

المقررة الإنتشار الأفقي والإيماس من غشاء بروتينات في الخلايا العصبية مثقف باستخدام الاسترداد الإسفار وPhotobleaching الإسفار الخسارة

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

الثقافات شريحة عضوي النمط لدراسة ديناميات دبقية قليلة التغصن وتكون الميالين

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

وتحليل وعائية عصبية إعادة عرض في Entorhino-الحصين الثقافات شريحة عضوي النمط

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

التحقيق في متشابك وسم / القبض على والصليب التقاط باستخدام الحاد شرائح الحصين من القوارض

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

عالية الدقة التحليل الكمي Immunogold من غشاء المستقبلات في الشبكية نقاط الاشتباك العصبي الشريط

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

أورجانوتيبيك شريحة هيبوكامبال الثقافات كنموذج للدراسة نيوروبروتيكتيون واختزاع الخلايا السرطانية

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...