Summary

إلى الوراء تحميل من الأعصاب، ومساحات، وجذور العمود الفقري مع الأصباغ الفلورية

Published: April 19, 2012
doi:

Summary

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

Abstract

وضع العلامات رجعي من الخلايا العصبية هي طريقة التشريحية مستوى 1،2 التي قد استخدمت أيضا لتحميل الكالسيوم والأصباغ الجهد حساسة إلى الخلايا العصبية 3-6. عموما، يتم تطبيق الأصباغ والبلورات الصلبة أو عن طريق الحقن الضغط المحلية باستخدام ماصات زجاجية. ومع ذلك، وهذا يمكن أن يؤدي إلى التخفيف من شدة العلامات صبغ وانخفاض، وخاصة عندما يطلب من عدة ساعات لنشر صبغ. هنا علينا أن نبرهن تقنية بسيطة ومنخفضة التكلفة لإدخال الأصباغ الفلورية وايون تراعي في الخلايا العصبية باستخدام ماصة شفط البولي ايثيلين مليئة الحل صبغ. هذا الأسلوب يوفر وسيلة يمكن الاعتماد عليها للحفاظ على نسبة عالية من الصبغة في اتصال مع محاور عصبية طوال الإجراء التحميل.

Protocol

وقد استخدمت dextrans فلوري كأدوات التشريحية وللتصوير نشاط الخلايا العصبية 1-4. الحقول وآخرون، (2009) 4 نشر بروتوكول لتطبيق ايون والجهد حساسة للأصباغ إلى مساحات محواري مع التركيز على الحبل الشوكي ونظام نموذجي. نحن هنا وصفا لإجراءات أكثر تفصيلا لتطبيق صبغة ال…

Discussion

We describe here a simple and cost-effective protocol for introducing dyes into neurons, nerves and spinal tracts. This method involves exposing identified anatomical pathways to a highly concentrated dye solution for the duration of the loading process. This results in retrograde labeling of the target site with little background compared to microinjections, bath application and electroporation techniques. However, the method is limited to sites in the nervous system where a tract of axons can be isolated. Furthermore,…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the intramural program of the National Institutes of Neurological Disorders and Stroke at the National Institutes of Health. We would also like to thank Dr. George Mentis for his earlier contributions to the method and to the data in Figure 3.

Materials

Material Name Company Catalogue number Comments
Tubing PE90 (IDxOD:0.034″x0.050″; Wall Thikness:0.008″) Clay Adams Brand Intermedic 427421 For Type I & Type II pipettes
NSF-51(IDxOD:1/16×1/8; Wall Thikness:1/32) PharMed BPT, Cole-Parmer AY242002 For Flexible tubing
Syringe 1ml insulin Syringe U-100 Becton Dickenson 329650  
Needle 19G x1-1/2″ Metal Hub Needle MONOJECT 200136 Connecting Type II pipette to syringe
  Alcohol Lamp      
Stopcock Three-way Stopcock with Male Luer Slip Adapter Baxter Healthcare Corp. 2C6241 Use with syringe
Holder H-1 electrode holder Narishige H-1/12  
Magnet stand Narishige GJ-8  
Micromanipulator Narishige M-3333  
Tweezers S&T DUMONT Swiss 00632-11 DUMONT JF-5 TC  

*Those are suggested materials. Can be replaced with any compatible products

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Cite This Article
Blivis, D., O’Donovan, M. J. Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes. J. Vis. Exp. (62), e4008, doi:10.3791/4008 (2012).

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