BakingTray
  • BakingTray Documentation
  • Getting Started
    • Hardware requirements
    • Known issues
    • Initial Installation
      • Software installation
      • Setting up ScanImage
    • Hardware setup
      • Motor Setup
        • PI stage setup
        • Calibrating a linear actuator
        • Verifying stage motions
      • Setting up a VT1000 vibratome
      • Setting up the laser
    • Finishing the install
      • Check the noise on your amplifiers
      • Starting BakingTray
      • Settings Files
      • Calibration
        • Basic calibrating procedures
        • Calibrating image size
        • Achieving high stitching accuracy
        • Fine-tuning positioning accuracy
        • Stitching tweak walkthrough
    • Stitching data
  • Users
    • Introduction
    • Sample preparation
    • User Guide
      • Starting BakingTray
      • Step 0: Loading the sample
      • Step 1: Setting imaging parameters
      • Step 2: Preparing the sample
      • Step 3: Selecting the imaging area
      • Step 4: Starting the acquisition
      • Step 5: Concluding the acquisition
      • Setting up checklist
      • Resuming an acquisition
      • Manual ROI acquisitions
    • Excitation choices
    • Choosing imaging settings
    • Troubleshooting
      • Hardware problems
      • Computer problems
      • Cutting problems
      • Imaging problems
    • Data structure
    • autoROI
  • Developers
    • Developers
      • Code overview
      • Developer notes
      • Motion control classes
      • The recipe file
      • Auto-ROI
      • Simulated mode
      • Contributing
    • FAQ
    • Gallery
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On this page
  • Perfusion
  • Mouse Perfusion
  • Rat perfusion tips
  • Protocols
  1. Users

Sample preparation

A major advantage of serial section microscopy is that it generates 3D images that are easy to register to a standard atlas and are particularly amenable to automated analyses. However, to take advantage of this the samples must be well prepared, undamaged, and well mounted. Samples that are damaged, full of blood, or mounted at extreme angles will be hard or impossible to work with later. Take sample preparation seriously!

Perfusion

Mouse Perfusion

  • Perfuse as normal in 4% PFA. Do not drop-fix. Do not use old PFA: opened PFA bottles can be kept for up to 3 months in the fridge.

  • Take great care removing the brain from the skull. Aim for zero damage. Use a dissection scope if it helps. Keep the olfactory bulbs and the cerebellum.

  • If you see dura adhering to the surface, remove it with care.

  • Post-fix overnight in 4% PFA at 4 degrees C. This is more important for rat brains: these may need two days.

  • Transfer to 1x PBS. Brains should sit in 1x PBS at least overnight before imaging as the higher osmolarity of 4% PFA will cause brains to expand once the enter the imaging buffer.

  • Sucrose is not involved: we aren't using a cryostat.

  • Longer term storage (months) is not encouraged because brains deform over time. If necessary, however, use 0.01% azide and label as such. Do not use higher azide concentration: that stuff is really dangerous.

Rat perfusion tips

For producing rat brains with all blood removed try the following:

  1. Use pentobarbital for overdose if you are not using it already, and use enough to knock it out in around 30s. About 2-3 ml per animal.

  2. Time between pento injection and starting perfusion should be as low as possible (under a minute). You will need a fast and precise dissection.

  3. Perfuse with about 150 ml of PBS or PB over 2-3 minutes followed by 200 ml of PFA for 4-5 minutes. The idea is to have enough flow at a good enough speed.

The basic cause of blood is more time spent after Pento injection and before start of perfusate flow leads to internal blood clotting (more viscous with time), and becomes difficult to push out especially in the finer vessels and capillaries. As for mouse brains, rat brains should sit in 1x PBS at least overnight before imaging as the higher osmolarity of 4% PFA will cause brains to expand once the enter the imaging buffer.

Protocols

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Last updated 2 months ago

50 mM PB slicing buffer
5% agar stock solution
single brain mounting protocol
Four brain embedding protocol