TITLE: Sectioning of Mouse Brain by Cryostat
AUTHORS: Maryana Nissan

[DESCRIPTION]
This protocol describes how to use the cryostat to prepare and slice mouse brain sections for Immunohistochemistry 

[STEPS]
SECTION: Preparation Methods
1. After removing the brain from -80C storage, leave the brain on dry ice for 15-20 minutes. This will ensure there is not a big temperature difference when it is placed in the chamber of the cryostat. The cryostat my lab uses is Leica, model CM-30505. 
SECTION: Preparation Methods
2. Label microscope slides. For embryonic brains, you will need between 8 to 10 slides. For early post-natal brains, you will need up to 10 slides and for adult brains, you will need 13-15 slides. 
SECTION: Preparation Methods
3. Set the chamber temperature (CT) and object temperature (OT). My lab often works with -24C for CT and -21C for OT. The temperature will take a few minutes to set. 
  

SECTION: Preparation Methods
4. When the temperatures of the cryostat are set, begin mounting the brain. 
SECTION: Preparation Methods
5. Place a disposable base mold [Fisher brand, CAT# 22-038-272] on the stand. 
SECTION: Preparation Methods
6. Fill half of the plate with embedding media 
SECTION: Preparation Methods
7. Gently transfer brain from dry ice to the center of the plate, securing it. Make sure to place it in the orientation you’d like. 
SECTION: Preparation Methods
8. Fill up the rest of the plate with embedding media. Note, when the embedding media freezes, it will turn white. This will make it difficult to tell the orientation of the brain. To prevent this, label the orientation of the brain on the plate. 
SECTION: Preparation Methods
9. Place the plate under the cold press and press it to cool. Leave the press on the plate for 2-5 minutes. 
SECTION: Preparation Methods
10. Lift the cold press, releasing the plate
SECTION: Preparation Methods
11. Remove the block from the plate. Now, you will have a frozen block. 
SECTION: Preparation Methods
12. Place the block on the concentric circle, and secure it with more embedding media
SECTION: Preparation Methods
13. Attach the concentric circle to the stand, and secure it by tightening the screw above it 
  

SECTION: Preparation Methods
14. Prior to slicing, double check the settings. Make sure you have the correct thickness 
SECTION: Slicing Methods
15. The cryostat can be run at an automated setting to section off the extra embedding media covering the brain. To do this, adjust the speed and thickness. Double press “RUN STOP” and “RUN ENABLE” to begin automated setting
  

SECTION: Slicing Methods
16. Adjust the settings of the stand as needed to ensure that the blade is slicing full sections of the brain
SECTION: Slicing Methods
17. Let the cryostat section the brain and occasionally stop it by double pressing “RUN STOP” and “RUN ENABLE” to brush off the embedding media on the stand 
SECTION: Slicing Methods
18. Stop the automated settings when you notice the cryostat has reached the brain. Begin manually collecting the brain sections. 
SECTION: Slicing Methods
19. Carefully mount each brain section onto a microscope slide by slightly tilting the slide over the brain section and pressing down. Be careful to not break the section, press down gently to lift the brain section. 
SECTION: Slicing Methods
20. Continue collecting sections until you have sliced the entirety of the brain
SECTION: Slicing Methods
21. Store slides at -80C