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MicroPunch Procedure in a RNase Free Environment Dong Ji, Jonathan Schultz, Bill McBride

MicroPunch Procedure in a RNase Free Environment Dong Ji, Jonathan Schultz, Bill McBride. Prepare 2-Methylbutane (Isopentane) / dry ice bath.

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MicroPunch Procedure in a RNase Free Environment Dong Ji, Jonathan Schultz, Bill McBride

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  1. MicroPunch Procedure in a RNase Free EnvironmentDong Ji, Jonathan Schultz, Bill McBride

  2. Prepare 2-Methylbutane (Isopentane) / dry ice bath. • Decapitate rat and quickly extract brain, flash freeze in isopentane for not more than 7 seconds. Remove brain, wrap in foil, and store at -80 C until ready to slice. • Using RNaseZap, clean all surfaces that will contact or be near the tissue including: all micro-punch needles, glass slides, interior surface of cryostat, cryostat blade, anti-roll bar, and all other tools that will be used.

  3. Acclimate brains and glass slides to cryostat set at -6 to -10 C for at least 2 hours. • Mount brains onto pedestals. • Sections will be cut at 300 um. If cryostat does not set that high, set at 60um and do 4 partial rotations of hand-wheel before 5th final cutting rotation to achieve desired 300 um section.

  4. Place up to 6 sections on a slide allowing sections to freeze mount to slide. • Store slides on dry ice until ready to do punches.

  5. Place frozen slide on Petri dish filled with dry ice to maintain temperature.

  6. Place dish and slide under dissection scope using cool fluorescent light as light source.

  7. Using a rat brain stereotaxic atlas, dissect out the areas of interest. Use only one dissection needle per region per rat (but can take from multiple sections before depositing tissue). Always use a needle diameter smaller than the region of interest. The sample does not need to contain the entire region, but should not contain tissue from outside the region of interest. • Chill punch needle on dry ice between each section to prevent tissue from thawing within the needle • Once all sections have been sampled, deposit tissue into 1.5ml Trizol in sterile tube. • Homogenize

  8. A dissection punch kit can be purchased from Fisher Scientific, but to eliminate cross contamination of RNase, each punch needle must be recleaned with RNaseZap between regions and animals. To avoid this repetitive (and time consuming) cleaning, we purchased thin walled acute cannula guides (Plastics One) of gauges 20, 19, and 18, which are respectively similar to punch diameters of 0.51, 0.77, and 0.98 mm. Each guide is fitted with a stylet to push out the collected tissue. The guides and stylets can be cleaned in bulk and enough can be prepared for an entire day of punching (i.e. separate guides and dummies for each region x animal). The guides can be assembled into a device to increase efficiency.

  9. Assembly procedure using Plastics One components The mounting holder is designed to hold a cannula guide straight along the length of the holder. However, side holding is needed for punching. A hole must be drilled in the side mounting holder to allow side holding of the cannula guide. Using a 1/8 inch drill bit, carefully drill through one side of the holder. The opposite side must remain intact to apply resistive force and hold the guide in place while punching. The hole should be drilled into the slot so that the stylet can be pushed through the slot on the opposite side and then through the cannula. The spring is placed on the stylet before being pushed through the slot. Once assembled, push down on the stylet to compress the spring, the stylet will project several mm beyond the end of the cannula. The stylet should be cut to length so that when compressed, only 0.5 mm of stylet projects beyond the end of the cannula (the stylet can be cut using a cutoff disk and Dremel). When the spring is then allowed to expand, there will be enough volume within the cannula to hold the tissue as it is being collected.

  10. Mounting holder showing intact slot and slot with hole drilled in it.

  11. Self-assembled device - components

  12. Final configuration of device

  13. If assembling a device is not viable, a prefabricated micropunch device can be purchased from Fisher Scientific. • Replacement punches can also be purchased separately from the kit (which contains 5 different needle sizes)

  14. Prefabricated device

  15. Part References • Fisher Scientific(www.fishersci.com) • Brain Tissue Punch Set, Fisher part number: NC9018224 • ~$400.00 per kit • Small Parts • Compression Spring, part number: B-CS-58 • 10 for $16.00

  16. Plastics One(www.plastics1.com) Prices vary upon quantity 18 Gauge thin wall acute guide cannula, part number: C309GATW; Stylet part number: C309DC 19 Gauge thin wall acute guide cannula, part number: C310GATW; Stylet part number: C310DC 20 Gauge thin wall acute guide cannula, part number: C311GATW; Stylet part number: C311DC Mounting Holder, part number: MH-300 Parts continued

  17. Additional Reading • IBRO Handbook Series: Methods in the Neurosciences Volume 2 • Brain Microdissection Techniques • Edited by A.C.Cuello Chapter 1

  18. Acknowledgements • Procedure developed by Dong Ji • Slides prepared by Jonathan Schultz joschult@iupui.edu

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