Cryostat - KravitzLab/KreedLabWiki GitHub Wiki

CryoStat

Goal:
Prepare histology slides for microscopic analysis.

Materials

  • Cryostat (reserve online)
  • Plastic molds
  • OCT
  • Brain sample in PFA
  • Filter paper
  • Cryostat blade
  • 1× PBS
  • 12-well plate
  • Dry ice (return to dry ice container or place in box to evaporate)
  • Paintbrushes
  • Glass slides
  • Glass slide covers
  • Stain (Fluoromount-G with DAPI)

Protocol

  1. Grab a mold (small cube) and add OCT (clear bottle with black cap) to the bottom. Mark the front of the mold.
  2. Take the sample and carefully pour off the PFA (corrosive) into the designated waste container under the sink.
  3. Use filter paper to dry the brain.
  4. Position the brain right-side up and place the anterior portion against the marked side of the mold. Ensure it is level.
  5. Fill the mold with OCT to approximately 2/3 full.
  6. Place the mold on dry ice and allow it to freeze for ~10 minutes or until opaque.
  7. Set the cryostat to –20°C and prepare a 12-well plate with 1× PBS in each well.
  8. Open the blade clamp using the right sidebar, insert the cryostat blade, tap it in using a paintbrush, and lock it in place.
  9. Adjust the knob on the right side of the sample holder to set the desired slice thickness. (Use anti-slip guard depending on slice size.)
  10. Remove the frozen block from the mold. Apply OCT to a chuck and attach the flat side of the sample so the anterior portion faces upward.
  11. Return the chuck with sample to the cryostat for 30 minutes to equilibrate.
  12. Set desired slicing parameters. Mount the chuck onto the cryostat. Ensure the blade is not too close to the sample to avoid breakage.
  13. Begin sectioning:
    • Identify brain landmarks during slicing using the brain atlas next to the machine.
    • To collect slices, slowly turn the knob while using a paintbrush to lift the edge. Pull gently as it turns, then transfer the slice using forceps into PBS.
      Label each well clearly to avoid confusion.
  14. Once sections are collected, bring them to the bench and prepare glass slides. Fill a larger well with 1× PBS.
  15. Cut the end of a pipette tip and use it to collect slices. Transfer slices into the PBS-filled well.
    • Submerge a glass slide in the well and arrange the sections in the same orientation using a paintbrush.
    • Label slides and allow them to fully dry.
  16. Add 100 μL of stain to each slide and place a glass cover slip on top.