VM, 5/7/12

Tissue Sectioning Protocol

Cryostat sectioning:
*Try to keep cover closed as much as possible to maintain temperature.
*Inside of cryostat is considered biohazard. Wear PPE gloves.
*Cryostat blade VERY sharp. Keep blade guard up when not actively sectioning.
Tools:
Paint brush
Razor blades

Cotton swab applicator sticks

Kimwipes & 100% EtOH spray bottle

Use 100% EtOH to decontaminate cryostat surfaces prior to use.

Freeze tissue to be sectioned:
1. Use tissue tech to create a platform on sectioning chuck slightly larger than the
width of the tissue.
2. Remove excess tissue tech surrounding tissue on popsicle stick.
3. Place desired tissue for section flat on chuck over tissue tech base. If tissue was
cut, apply tissue tech to the exposed ends of the remaining tissue to prevent
fracture.
4. Cover with tissue tech to surround the tissue and place chuck on freezing bay.
*Be sure to remove bubbles in tissue tech.
5. Press Freeze Object button on cryostat and watch for tissue tech to solidify.
Mounting on cryostat:
1. Remove chuck from bay and attach to mounting apparatus.
-Unscrew knob and ensure chuck is pushed all the way into mount.
2. Adjust chuck angle with blade using right lever to unlock the mount and knob to
adjust chuck.
3. Use retract and advance arrow buttons to bring the chuck close to the blade.
- Press and hold down arrow to move chuck towards blade.
- Up arrow will continue to retract chuck until pressed again.
4. Once chuck is relatively close to blade, unlock wheel moving pin towards you and
begin advancing the chuck closer spinning the wheel.
*The trim button will flip the distance advanced/rotation between 50 microns and 10
microns.
Sectioning:
1. Continue cutting through tissue tech layer until tissue to be sectioned appears.
2. When ready to take a section,
- Use brush to remove excess debris.
- Wipe off platform and polycarbonate guard.
- Use cotton swab to clean blade with strokes moving from platform to the
outside tip of blade.
3. Place polycarbonate guard down flat on platform.

VM, 5/7/12
4. Turn wheel in a controlled, uniform motion once in contact with tissue.
Section should then slide in between platform and guard.
-If section folds, reset the apparatus and re-clean the blade and platform
surfaces.
* Optimal operating temperature is -27 to -29C. If tissue continues to fold or
not section cleanly, temperature may be out of range. Adjust temp and wait
for it to change in the cryostat.
-Dull blades are also a culprit for poor sectioning. Blades are mounted so that
the actual cutting area is moved from right to left. Replace blade if it is
completely used.
5. With section on platform, hover slide over section moving from one side to the
other to allow it to adhere to slide without creating air bubbles. (2 sections per slide)
Cleaning up:
Remove trap being sure not to allow shavings to fall from trap into machine and
discard into trash.
Clean and replace trap.
Wipe down all surfaces with 100% EtOH.

OEM Slide preparation protocol:

1. Snap freeze fresh tissues in liquid nitrogen or isopentane pre-cooled in liquid
nitrogen, embedded in Tissue-Tech OCT (PS002) compound in cryomolds. Store
frozen blocks at - 80 C.
2. Cut 4-8 um thick cryostat sections (protocol below) and mount on superfrost plus
slides or gelatin coated slides. Store slides at - 80 C until needed. The slides can be
stored at -20 C for short term storage (within a few weeks).
3. Before staining, warm slides at 37 for few minutes.
4. Fix the sections in 95% ethanol for 5 minutes.
5. Transfer to 70% ethanol for 5 minutes.
6. Transfer to 40% ethanol for 5 minutes.
7. Transfer to distilled water for 1 minutes, air dry.
8. Transfer to 40% ethanol for 3 minutes.
9. Transfer to 70% ethanol for 3 minutes.
10. Transfer to 95% ethanol for 3 minutes.
11. Store slides at 4 and proceed to standard staining procedure.