Buck Institute Morphology Microtome Protocol

 Return the chuck holder to home position.

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

Turn on microtome, make sure all locks are set and blade guard is in place.

Have tools accessible - forceps, brushes, kimwipes, blades, ice water.

Place water bath near microtome. Water temperature 40-45 C

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.

Set the sectioning thickness. Usual settings are 5-7 microns for sectioning, 10+ microns for trimming.

Paraffin blocks should be cold for optimum sectioning.

Lock the handwheel before installing the paraffin block into

the specimen holder. Open the clamp and install the block.

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.

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.

At this point you can trim the paraffin block to expose the

sample.

Trim until you have the sample orientation correct and are getting complete sections per cycle.

Sections are cut gently with a uniform, slow rotation. Adjust your speed so that you can safely receive the sections.

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.

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.

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.

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.

Collect the amount of sections required for each slide.

Drain the slide of water by placing vertically for several minutes

After sectioning is completed, lock the flywheel and remove the block from the specimen holder.

Return the chuck holder to home position

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.