TITLE: Buck Institute Morphology Microtome Protocol
AUTHORS: Stella Breslin, Akos A Gerencser

[DESCRIPTION]
Buck Institute Morphology uses automatic rotary microtomes to section paraffin embedded tissue. 
Safety considerations must be followed to safely acquire paraffin sections on an automatic microtome. The following protocol will describe steps to successfully and safely use the microtome for paraffin sections on glass slides.


[STEPS]
SECTION: Microtome setup
1. Turn on microtome, make sure all locks are set and blade guard is in place. 



SECTION: Microtome setup
2. Have tools accessible - forceps, brushes, kimwipes, blades, ice water.
Place water bath near microtome. Water temperature 40-45 C

SECTION: Microtome setup
3.      
Set up blade holder angle between 5 to 10 degrees. Replace the blade if needed. Do not have the blade holder directly under specimen holder when installing the block. Orient yourself with each of
the clamps that hold the blade holder in position. Make sure they are tight (do not overtighten) before starting to section. It is dangerous to trim or sectionif the knife holder is not secure.
4. Set the sectioning thickness. Usual settings are 5-7 microns for sectioning, 10+ microns for trimming.
5.  Paraffin blocks should be cold for optimum sectioning.
SECTION: Install the paraffin block
6. Lock the handwheel before installing the paraffin block into
the specimen holder. Open the clamp and install the block.
SECTION: Install the paraffin block
7. Orient the block so that block face travels parallel to the
blade by using the adjustment screws on the specimen holder. This can be
refined when the sample is close to the blade. This adjustment is to correctly
position the block for optimal sectioning. 
SECTION: Install the paraffin block
8. Carefully bring the block to the blade. You can manually
rotate the handwheel or use the automatic functions. When the block touches the
blade, you can slowly rough cut and adjust until an entire section is produced
per rotation.
SECTION: Install the paraffin block
9. At this point you can trim the paraffin block to expose the
sample.
SECTION: Sectioning
10. Trim until you have the sample orientation correct and are getting complete sections per cycle.
SECTION: Sectioning
10.1. Sections are cut gently with a uniform, slow rotation. Adjust your speed so that you can safely receive the sections.
SECTION: Sectioning
10.2. Ribbons can be created with an even speed of rotation. You can use tweezers or brushes to help the ribbon stay down on the blade holder.
SECTION: Sectioning
10.3. Between rotations some samples like liver require “soaking” Carefully, briefly place a damp Kim wipe on the sample before the rotation starts downward. Slow the speed to do this safely.
SECTION: Water Bath section flotation
11. Bring your sections from the blade holder to the water bath and slowly lower your ribbon into the water. With a smooth motion you can avoid getting air bubbles or wrinkles. 

SECTION: Water Bath section flotation
12. Take a clean positively charged glass slide and slide it gently under the sections. Slowly raise it to connect the section to the slide, minimizing trapping air bubbles between sample and slide.
SECTION: Water Bath section flotation
13. Collect the amount of sections required for each slide.
SECTION: Water Bath section flotation
14. Drain the slide of water by placing vertically for several minutes 
SECTION: Remove blocks
26. 
SECTION: Remove blocks
14.1. After sectioning is completed, lock the flywheel and remove the block from the specimen holder.
SECTION: Remove blocks
0.26.      Return the chuck holder to home position.
SECTION: Remove blocks
0.26.    If all sectioning has been completed, clean the equipment of wax. Remove disposable blade to used blade holder or sharps container.  Clean water bath if needed
SECTION: Remove blocks
15. Return the chuck holder to home position

SECTION: Remove blocks
16. If all sectioning has been completed, clean the equipment of wax. Remove disposable blade to used blade holder or sharps container.
SECTION: Remove blocks
17. Clean water bath if needed.