Jun 13, 2023
Sectioning of Mouse Brain by Microtome
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: maryana.nissan 2023. Sectioning of Mouse Brain by Microtome. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbxd91lpk/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: June 13, 2023
Last Modified: June 01, 2024
Protocol Integer ID: 83281
Keywords: ASAPCRN, mouse brain by microtome, slice mouse brain sections for immunohistochemistry, microtome, slice mouse brain section, immunohistochemistry, mouse brain, mouse
Funders Acknowledgements:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: ASAP-020600
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
This protocol describes how to use the microtome to prepare and slice mouse brain sections for Immunohistochemistry
Preparation Methods
While preparing the microtome, make sure the blade is covered. The microtome used in the lab is from Leica Microsystems.
Slide the plate into the stage and secure it by tightening it with a screw.
Orient the plate so that it is centered.
Microtome setup, with the plate attached.
Add a few drops of 90% of ethanol on the sides of the plate and place dry ice to cool the microtome. The microtome will be cooled and ready for usage when the plate accumulates ice.
When the microtome is cooled, add a big drop of 1X Phosphate-Buffered solution [1X PBS] to the center of the plate
Quickly orient the brain on top of the 1X PBS bubble, ensuring that it is centered.
Double check the brain is in the center and straight. When it is, add more 1X PBS to secure it.
Add powdered dry ice over the brain to cool it. Let the brain cool for 2-5 minutes prior to slicing.
Prior to slicing, adjust the thickness setting. This knob is found on the side of the microtome.
Prepare the 24-well culture plate (Thermo Scientific, CAT # 142475] with cryoprotectant fluid. Brain sections will go here after they are sliced.
Slicing Methods
Remove the protective cover of the blade.
Move the stage up, placing the brain just below the blade.
Slowly move the blade towards you, slicing a section of the brain. To prevent the sections from folding and breaking, slice slowly and in a single continuous motion.
Gently scoop the brain section away from the blade, using a thin painting brush.
Slowly place the brush into cryoprotectant fluid. Gently move the brush to allow the section to unfold in the liquid.
Press the button on the side of the microtome to push the stage 1 point higher. This will allow you to slice the next brain section, at the thickness you have set.
Continue slicing until you have sliced the entirety of the brain.
Secure the 24-well plate with tape. Store brain sections in cryoprotected fluid at -20C.