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