Jun 12, 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 12, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 83281
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: ASAP-020600
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Abstract
This protocol describes how to use the microtome to prepare and slice mouse brain sections for Immunohistochemistry
Preparation Methods
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
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.