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:
- Fixation:
- Immerse tissue in 4% PFA at 4°C. Fixation time is sample-dependent (see immunostaining protocol)*
- Rinsing:
- Remove PFA and rinse with 1X PBS.
- 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.
- 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:
- 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.
- 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.
- 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.
- 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:
- 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.
- 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).
- 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.
- Collecting Sections:
- Drying and Storage of Slides:
- Allow sections to air-dry briefly at room temperature
Staining
- Incubate with TBS + 0.1% triton (TBST) for 15 minutes to remove OCT3.
- Wash with TBST 5 minutes x 3 times at room temperature
- Incubate samples w/ blocking buffer for 1 hour at RT
- Block Buffer: 10% donkey serum
- Spin primary antibody for 1hr at max speed while sample is blocking
- Prepare primary antibody in blocking buffer
- 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.
- Wash in TBST for 5 minutes x 3 times at room temperature
- Prepare secondary antibody solutions in blocking buffer * Spin diluted secondary antibody for 1 hr at max speed
- Add secondary antibody solution and incubate samples overnight at 4C followed by 1hr at RT
- Wash samples in TBST for 5 minutes x 3 times at RT
- Mount samples in Flouromount G