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

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

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• 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.

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• 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.

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Place frozen slide on Petri dish filled with dry
ice to maintain temperature.
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Place dish and slide under dissection scope using
cool fluorescent light as light source.
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• 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

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• 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.

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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.

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Mounting holder showing intact slot and slot with
hole drilled in it.
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Self-assembled device - components
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Final configuration of device
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• 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)

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Prefabricated device
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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

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Parts continued

• 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

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Additional Reading

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

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Acknowledgements

• Procedure developed by Dong Ji
• Slides prepared by Jonathan Schultz
  joschult_at_iupui.edu