TITLE: Sectioning Mouse Brain with Sliding Microtome
AUTHORS: Naveen Ouellette, daphne.toglia, Holly Myers

[DESCRIPTION]
This is a protocol that details sectioning a mouse brain fixed in 4% PFA and 30% sucrose on a sliding microtome. This protocol details the microtome set up, the mounting of the brain on the microtome stage in OCT or 30% sucrose, slicing the brain with the microtome blade, microtome take down and clean up, and tissue section storage.

[BEFORE_START]
The mouse brain must be fixed in 30% sucrose for 1-2 days prior to sectioning on microtome, or until the brain sinks to the bottom of the container of 30% sucrose.

[STEPS]
SECTION: Setup
2. Microtome setup:
SECTION: Setup
2.2. Ensure lock handle for knife is in the closed position before making any adjustments to, or mounting tissue onto, the microtome.

 


 

 

SECTION: Setup
2.3. Attach the stage to the microtome and ensure that it is level.
 

SECTION: Setup
2.4. Take microtome knife out of its case and use 70% ethyl alcohol and a Kimwipe to remove the protective oil coating on the knife.

Insert knife into the holder and tighten both black-knobbed clamps to hold knife in place. Black knife guard located below knobs can be used to cover blade when microtome blade is inserted but not in use.

 

 

SECTION: Setup
2.5. Set the knife angle to desired setting with knife angle adjustment knob. For example, 0 degrees.

 

SECTION: Setup
2.6. Adjust the section thickness in microns by turning the section thickness adjustment dial.

 

 


SECTION: Setup
3. Create a base of 1xPBS, OCT, or 30% sucrose on the microtome stage:

 

SECTION: Setup
3.1. Fill the trough surrounding the stage with dry ice chunks.

 

SECTION: Setup
3.2. Wait until stage has cooled enough so that the 1xPBS, OCT, or sucrose will freeze soon after it is applied. The metallic stage should look slightly white and frosty, and might take 5 min to reach this point.

SECTION: Setup
3.3. Apply a 4cm square layer of 1xPBS on the stage to section one brain.

 


 
 

SECTION: Setup
3.4. Wait until the 1xPBS is completely frozen before proceeding.
SECTION: Setup
3.5. Determine if you will mount the specimen in OCT or sucrose. If mounting in OCT, proceed to step 4. If mounting in sucrose, proceed to step 5.

 

SECTION: Mounting specimen using OCT
4. Mount the specimen on microtome stage using OCT:

 

SECTION: Mounting specimen using OCT
4.1. With a razor blade, trim tissue as needed so it will sit flat and level on the stage in the correct orientation (coronal, sagittal) when sectioning.
SECTION: Mounting specimen using 30% sucrose
5. Mount the specimen on microtome stage using 30% sucrose:

 

SECTION: Mounting specimen using OCT
4.2. Blot specimen briefly on a Kimwipe to remove excess liquid.
SECTION: Mounting specimen using OCT
4.3. Add a large drop of OCT onto the frozen, flat OCT base.
SECTION: Mounting specimen using OCT
4.4. Quickly set the specimen into the large drop of OCT before it freezes.
SECTION: Mounting specimen using OCT
4.5. Gently hold the tissue in place until the OCT has solidified enough to hold the tissue securely.
 

SECTION: Mounting specimen using OCT
4.7. Wait until tissue freezes passively, about 1 min , and gently brush away crushed ice to reveal brain specimen. The tissue should be hard and white after freezing.

SECTION: Mounting specimen using OCT
4.6. Crush dry ice with a rubber mallet and apply ice dust to the top of the mounted brain.
 

SECTION: Mounting specimen using 30% sucrose
5.1. With a razor blade, trim tissue as needed so it will sit flat and level on the stage in the correct orientation (coronal, sagittal) when sectioning.
SECTION: Mounting specimen using 30% sucrose
5.2. Blot specimen briefly on a Kimwipe to remove excess liquid.
SECTION: Mounting specimen using 30% sucrose
5.3. Add a large drop of 30% sucrose onto the frozen, flat 30% sucrose base.
SECTION: Mounting specimen using 30% sucrose
5.4. Quickly set the specimen into the large drop of sucrose before it freezes.
SECTION: Mounting specimen using 30% sucrose
5.5. Surround base with more sucrose until you have a sturdy base that will hold the specimen securely.  Add slowly drop by drop and avoid making hollow cavities.
SECTION: Mounting specimen using 30% sucrose
5.6. Keep adding sucrose until specimen is securely held or if preferred, completely
covered with sucrose.

 

SECTION: Sectioning
6. Section the tissue:
SECTION: Sectioning
6.2. Move the platform and specimen vertically towards the knife by turning the coarse feed wheel located on the left side of the microtome. 
SECTION: Sectioning
6.3. To position blade as close as possible to the specimen, pull the manual feed lever forward to raise the platform by microns. 
SECTION: Sectioning
6.4. Pull the knife sledge movement grip forward to bring the knife through the tissue. 
SECTION: Sectioning
6.5. The tissue section will remain on the top of the knife. Wrinkles and deformation of the tissue is normal.
SECTION: Sectioning
6.6. Pick up the section with a fine-tipped paintbrush. Move each section to the well plate filled with 1xPBS & Sodium Azide 0.01% .


 


SECTION: Sectioning
6.7. If needed, wipe the blade with a Kimwipe to clear any excess moisture or debris.
SECTION: Sectioning
6.8. Push the knife sledge movement grip back to bring the blade back to the starting point. 
SECTION: Sectioning
6.9. Pull the manual feed lever forward to raise the specimen for the next section.
SECTION: Sectioning
6.11. Repeat steps 6.4 - 6.10 until desired sections are collected. Tissue may be double or triple stacked in each well in the well plate, but ideally sections in the same well should be separated by at least a few hundred microns (ex: every 6th section or more) so it is easier to see anatomical differences and determine section order when mounting.
SECTION: Sectioning
6.12. Wrap well plate in parafilm and secure with tape to prevent spillage, then wrap in aluminum foil to protect from light, and store at 4 °C until ready to stain the tissue and mount the sections onto slides. Tissue may be stored for several months.
SECTION: Clean up
7. Take down microtome and clean up equipment:
SECTION: Sectioning
6.1. If desired by requestor, use a razor blade to cut a shallow notch on either the left or right side of the cortex of the brain that runs posterior to anterior. Make a note of which side is notched and refer to it for left/right orientation when mounting sections on slides.
SECTION: Clean up
7.1. Spray the knife and microtome with 70% ethyl alcohol and carefully wipe dry with Kimwipes.

SECTION: Clean up
7.2. 
Coat the knife with mineral oil and carefully store in its case.

SECTION: Clean up
7.3. Detach the specimen stage and run warm tap water over it to release 1xPBS, OCT, or 30% sucrose and any residual tissue.
SECTION: Clean up
7.4. Based on requestor preference, either retain tissue or dispose of tissue in biohazard waste bin.
SECTION: Clean up
7.5. Spray the stage with 70% ethyl alcohol. Soak for 3 min  before wiping with a Kimwipe.
SECTION: Clean up
7.6. Dry the stage thoroughly to prevent frost buildup.
SECTION: Mounting specimen using OCT
4.8. Proceed to step 6 for sectioning.
SECTION: Sectioning
6.10. Add more dry ice as needed to the troughs in the stage to prevent the OCT or sucrose mounting solution from softening or melting, as the dry ice added initially will melt over time. 
SECTION: Setup
1. 
Work station setup:

SECTION: Setup
1.1. Set up a well plate next to the microtome. This will be used to store the brain sections that are sliced during this protocol. Wells should be filled with 1xPBS & Sodium Azide 0.01% for tissue preservation. Label the well plate lid with relevant information: brain ID number, section thickness, date, etc.


 

SECTION: Setup
1.2. Set up a dish filled with 1xPBS next to the microtome to collect any miscellaneous sliced tissue that will be discarded and not saved for storage.
SECTION: Setup
2.1. Microtome diagrams with labeled parts: