Dec 14, 2023

Public workspaceSerial Sectioning of Mouse Brain

DOI
dx.doi.org/10.17504/protocols.io.dm6gp3x78vzp/v1
  • 1University of Alabama at Birmingham, Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics
Jhodi Webster
Open access
DOI: dx.doi.org/10.17504/protocols.io.dm6gp3x78vzp/v1
Protocol Citation: Asta Zane, Nicole J Corbin-Stein, Gabrielle Childers, Jhodi Webster, Vickie Yang, Woong-Jai Won, Rajesh Gupta, Ashley Harms 2023. Serial Sectioning of Mouse Brain. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3x78vzp/v1
Manuscript citation:
Williams GP, Schonhoff AM, Jurkuvenaite A, Gallups NJ, Standaert DG, Harms AS. CD4 T cells mediate brain inflammation and neurodegeneration in a mouse model of Parkinson's disease. Brain. 2021 Aug 17;144(7):2047-2059. doi: 10.1093/brain/awab103. PMID: 33704423; PMCID: PMC8370411.

Schonhoff, A.M., Figge, D.A., Williams, G.P. et al. Border-associated macrophages mediate the neuroinflammatory response in an alpha-synuclein model of Parkinson disease. Nat Commun 14, 3754 (2023). https://doi.org/10.1038/s41467-023-39060-w
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: November 03, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 90385
Keywords: ASAPCRN
Funders Acknowledgement:
Co-pathologies Drive Neuroinflammation and Progression in PD
Grant ID: ASAP-021030
Abstract
This protocol highlights sectioning 40 micron mouse brain tissue to be used for histology experiments.
Guidelines
Make sure to punch the hole in the non-injected side of the brain. That will allow to quantify the staining much easier later. If injected bilateral, then do not punch the hole. The hole location will be different for different parts of the brain: for striatum it will be in the cortex, and for SNpC in the middle.
Materials
 
  •       Sliding microtome HM450 (Thermo Scientific)
  •       Microtome blades (Thermo Scientific , e.g. Epredia MX35 Premier Microtome Blades)
  •       Microtome metal stage
  •       Two buckets of dry ice with cover (one for crushed dry ice, and one for the regular dry ice)
  •       Dry ice
  •       Foil
  •       Paint brush
  •       Large Petri dish with phosphate  Buffered Saline (PBS) 
  •       30% sucrose
  •       50% glycerol in phosphate buffered saline (PBS) (antifreeze solution)
  •       24 or 12 well plates ( for sectioning 1/6 or 1/12 of the brain)
PROCEDURE
PROCEDURE
Label 24 or 12 well plate with appropriate identifiers (date, project, region to be sectioned etc.) and fill each well ⅔ full with anti-freeze solution (50% glycerol in PBS).
Freeze microtome stage on the dry ice.
Crush dry ice for the sprinkling the stage later
When ready place the stage into place, level it. Put the dry ice into it to keep it cold while working. 
Put the microtome blade into the place, keep it covered not to get injured.
Place few drops of sucrose on the cold stage and put your brain on it. The brains bottom will be hindbrain, and the top of the brain will be the forebrain. The brain lateral side will be facing you, and dorsal away from you. Put few more drops of sucrose to support the brain. Sprinkle crushed dry ice around the brain and cover the whole stage with foul for 2-3 min, to fully freeze.
Prior to sectioning, adjust the thickness setting, all sections will be 40 microns thick, and trimming has to be 80 microns. This knob is found on the side of the microtome.
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 antifreeze solution. Gently move the brush to allow the section to unfold in the liquid. Make sure the sections go into appropriate wells.
Continue slicing until you have sliced the entirety of the brain.
Secure the well plate with tape. Store brain sections in antifreeze solution at -20C.