Sep 29, 2023
Buck Institute Morphology Microtome Protocol
- 1Buck Institute for Research on Aging
Protocol Citation: Stella Breslin, Akos A Gerencser 2023. Buck Institute Morphology Microtome Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkodrdv5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 27, 2023
Last Modified: September 29, 2023
Protocol Integer ID: 88497
Keywords: Micronomy, Microtome, Sections, paraffin blocks, histology, slide
Abstract
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.
Materials
Morphology Microtome RM2255 -automatic rotary microtome
Morphology Microtome RM2155 -automatic rotary microtome
Epredia HM 355 S Microtome E System
Water Bath
Sakura AccuEdge 4689 Low Profile Blade
Safety warnings
The microtome uses sharp disposable blades. Use caution when removing and installing blades. Always have the handwheel locked when manipulating the blade holder. Fully tighten the blade holder before attempting to section.
Microtome setup
Microtome setup
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.
Install the paraffin block
Install the paraffin block
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.
Sectioning
Sectioning
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.
Water Bath section flotation
Water Bath section flotation
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
Remove blocks
Remove blocks
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.
Protocol references
Prophet, Edna B. “Chapter 8 Micotomy.” Laboratory Methods in Histotechnology, American Registry of Pathology, Washington, D.C., 1992.
101 STEPS TO BETTER HISTOLOGY, Leica BioSystems