Cryo-Embedding and Slicing

Reference: Goodwin, Katharine et al. “Plasticity in airway smooth muscle differentiation during mouse lung development.” Developmental Cell, Volume 58, Issue 5, 2024, pp. 338-347.e4.

You’ll need

  • 4% Paraformaldehyde (PFA)
  • Phosphate Buffered Saline (PBS)
  • Tris Buffered Saline (TBS)
  • OCT Compound
  • 20% Sucrose in 1X PBS: (2g to 10 mL). Store at 4°C.
  • 30% Sucrose in 1X PBS
  • 1:1 OCT:30% Sucrose Solution. Store at 4°C.
  • Embedding Molds
  • Dry Ice
  • Microtome Blades
  • Fine Paintbrush (teeny tiny, like for fly work or painting minatures)
  • Frosted glass Coverslips
  • Paper Towels
  • Triton X-100
  • Wash Buffer (TBST): TBS + 0.1% Triton X-100
  • Blocking Buffer: 10% Donkey Serum in TBST.
  • Fluoromount-G

Tissue Fixation and Cryoprotection

Procedure:

  1. Fixation:
    • Immerse tissue in 4% PFA at 4°C. Fixation time is sample-dependent (see immunostaining protocol)*
  2. Rinsing:
    • Remove PFA and rinse with 1X PBS.
  3. Cryoprotection:
    • Transfer samples to 20% sucrose/PBS solution.
    • Incubate on a shaker at room temperature for at least 1 hour. Tissue sinking indicates adequate sucrose infiltration.
    • Transfer samples to 30% sucrose/PBS solution.
    • Incubate on a shaker at room temperature for at least 1 hour. For denser or larger tissues, this step can be overnight.
  4. OCT Infiltration:
    • Transfer samples to the 1:1 OCT:30% sucrose solution.
    • Incubate on a shaker at room temperature for 1 hr.
    • Transfer samples to fresh 1:1 OCT:30% sucrose solution.
    • Incubate on a shaker at 4°C overnight. This ensures complete infiltration with the embedding medium.

Tissue Embedding and Freezing

This process involves orienting the cryoprotected tissue in OCT within a mold and rapidly freezing it.

Procedure:

  1. Prepare Molds and Workspace:
    • Label molds clearly with the permanent marker.
    • Place a small amount of OCT in a layer at the bottom of each mold.
  2. Transfer and Orient Tissue:
    • Transfer the tissue into the OCT-lined mold.
    • Gently orient the tissue to the desired cutting plane. This is a critical step for getting nice sections. * If the tissue is small, you can transfer it with a small bubble of the OCT/sucrose solution.
    • Carefully pipette or wick away any excess 1:1 OCT:30% sucrose solution from around the tissue within the mold.
  3. Add OCT and Initial Freeze:
    • Remove one piece of dry ice, maybe 1-2 inches long, and place on bench to sublimate until it has a flat surface.
    • Flip the dry ice piece over and place a plastic cube insert onto the flat surface. After a few moments the OCT will turn white as it freezes.
  4. Complete Embedding and Freezing:
    • Once the base layer with the tissue is partially frozen and stable, add OCT to cover the tissue and fill the mold. Avoid air bubbles.
    • Hold the mold on the dry ice until the entire OCT block is opaque white and frozen solid. This may take several minutes depending on the block size.
    • Store in -80.

Slicing

Cryostat Preparation:

  • Ensure the cryostat chamber is at -21°C. If the cryostat is warm, it may be someone is intentionally deforsting it

Slicing Procedure:

  1. Prepare Workspace and Mount Block:
    • Place paper towels or absorbent paper in the cryostat chamber below the blade holder to catch trimmings.
    • Securely install a razor blade into the blade holder.
    • Use the OCT like glue at this temperature. Apply a thin layer of fresh OCT to the surface of a pre-cooled specimen chuck.
    • Quickly press your frozen OCT block onto the OCT on the chuck. Allow it to freeze firmly in place for a few minutes.
  2. Mount Chuck and Align Block:
    • Mount the chuck with the sample block into the cryostat’s specimen holder.
    • Coarsely trim the block using the cryostat’s advance mechanism until the blade is near the ice.
    • Adjust the orientation of the block relative to the blade to ensure the cutting face is parallel to the blade edge.
    • Try a test slice, the blade will hit one corner first, re-adjust the orientation and repeat until hitting all four corners of the sample block.
    • Coarse cut until you reach your sample
    • Set the desired section thickness (typically ~10 µm).
  3. Sectioning and Collection:
    • Collecting Sections:
      • With Plate: If using a plate, adjust it to be parallel and very close to the blade edge. As the section is cut, it should slide between the blade and the plate.
      • With a Paintbrush:
        • As the section begins to come off the blade, use a thin paintbrush held at a low angle to gently guide the leading edge of the section and prevent it from curling.
    • Transferring to Slide:
      • Carefully bring a room-temperature glass slide towards the section.
      • Align the section with the slide and gently touch the slide to the section. The section will adhere to the slide.
  4. Drying and Storage of Slides:
    • Allow sections to air-dry briefly at room temperature

Staining

  1. Incubate with TBS + 0.1% triton (TBST) for 15 minutes to remove OCT3.
  2. Wash with TBST 5 minutes x 3 times at room temperature
  3. Incubate samples w/ blocking buffer for 1 hour at RT
    • Block Buffer: 10% donkey serum
  4. Spin primary antibody for 1hr at max speed while sample is blocking
    • Prepare primary antibody in blocking buffer
  5. Add primary antibody and incubate overnight at 4 C followed by 1 hr at RT (or at least 1 hr RT)
    • You can store samples in an old pipetting box, with wet paper towels underneath to help evaporation.
  6. Wash in TBST for 5 minutes x 3 times at room temperature
  7. Prepare secondary antibody solutions in blocking buffer * Spin diluted secondary antibody for 1 hr at max speed
  8. Add secondary antibody solution and incubate samples overnight at 4C followed by 1hr at RT
  9. Wash samples in TBST for 5 minutes x 3 times at RT
  10. Mount samples in Flouromount G