Aug 18, 2022

Public workspacePreparation and cryosectioning of fixed mouse brains

DOI
dx.doi.org/10.17504/protocols.io.3byl4bmeovo5/v1
  • 1The University of Sydney, Sydney, Australia
Benjamin Trist
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DOI: dx.doi.org/10.17504/protocols.io.3byl4bmeovo5/v1
Protocol Citation: Benjamin Trist, Courtney Wright, Alejandra Rangel 2022. Preparation and cryosectioning of fixed mouse brains. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4bmeovo5/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: August 12, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 68565
Keywords: NCAM, HNA, Human-to-mouse xenograft, Human iPSC, Immunohistochemistry, ASAPCRN
Funders Acknowledgement:
Michael J Fox Foundation
Grant ID: ASAP-000497
Abstract
This protocol describes how to prepare thin, fixed mouse brain tissue sections from whole fixed mouse brains in preparation for immunohistochemistry or histology. This process includes immersion fixation and cryoprotection of mouse brains, followed by flash freezing of whole brains and cryosectioning into thin tissue sections using a freezing microtome.
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Guidelines
Contributors:
1.ASAP Teams (e.g. Kirik, Alessi, Scherzer, Lee).
a.Kirik.
2.Labs (e.g. Kirik, Parish, Thompson, Halliday, Sue, Johnston).
a.Kirik.
Materials
Equipment:

  • Small metal ramekin
  • Tweezers
  • Vertical rocker
  • Freezing microtome

Consumables:

  • Amount5 mL , Amount50 mL sample containers
  • Dry ice
  • Foil
  • Paint brushes
  • Microtome blades (e.g. Epredia MX35 Ultra Low Profile Microtome Blades)

Key reagents:

  • Isopentane
  • Sodium azide
  • Sucrose
  • Optimal Cutting Temperature (OCT) compound

Solutions:

  • 10x PBS
1. Amount77.3 g of NaH2PO4.H2O (Concentration0.28 Molarity (M) ), Amount203.7 g of Na2HPO4 (Concentration0.72 Molarity (M) ), Amount177.4 g of NaCl (Concentration1.5 Molarity (M) ) in Amount2 L dH2O, pH 6.9
  • 1x PBS, pH 7.4
1. Amount100 mL of 10x PBS in Amount900 mL dH2O, no pH adjustment required
  • 30% sucrose in 1x PBS
1. Amount300 g sucrose up to Amount1 L with 1x PBS
  • Anti-freeze solution
1. Amount300 mL (30%) glycerol, Amount300 mL (30%) ethylene glycol up to Amount1 L with 1x PBS

Material input (animal, cell, tissue, fraction details)

Whole mouse brains fixed by transcardial perfusion with 4% paraformaldehyde, stored overnight following perfusion in 50mL containers containing 4% paraformaldehyde.
Cryoprotection and snap freezing
Cryoprotection and snap freezing
Remove fixed brain from 50 mL sample container and discard 4% paraformaldehyde solution into appropriate waste disposal stream.
Place brain back into the Amount50 mL sample container and fill with 30% sucrose in 1x PBS containing 0.01% sodium azide.
Place container on vertical rocker at Temperature4 °C for Duration48:00:00 or until brain no longer floats in sucrose solution.
2d
Half fill small metal ramekin (~Amount100 mL capacity) with isopentane (2-methyl-butane) and place on dry ice, allowing it to cool to Temperature-45 °C .
Use plastic tweezers to remove brain from sucrose and immerse in isopentane solution for Duration00:00:15 -Duration00:00:20 .
  • Monitor solution temperature to ensure it does not drop below Temperature-60 °C , as temperatures lower than this can cause tissue to crack and fragment.
  • Use tweezers lacking teeth, as these will damage/mark the cortex.
35s
Remove frozen mouse brains and either'
  • Section immediately, or
  • Wrap in foil, then wrap in tissue paper, then place in labelled sample tube Temperature-80 °C for storage.
Serial cryosectioning of grafted mouse brains for thin tissue histology
Serial cryosectioning of grafted mouse brains for thin tissue histology
If brain tissues are stored at Temperature-80 °C , move them to Temperature-20 °C DurationOvernight prior to sectioning.

Overnight
Label 12 well microtiter plates with appropriate identifiers (brain tissue IDs, series #s, etc.) and fill each well ⅔ full with anti-freeze solution.
Cool freezing microtome sample holder with dry ice.
Squeeze OCT onto microtome specimen head until the diameter is approximately the diameter of a Thikness1.5 cm piece.
Allow OCT to freeze (turns opaque from translucent) then shave layers off by cutting with microtome blade until you have created a flat base, the thickness of which should be a little more than Thikness4 mm -Thikness5 mm .
Mount caudal end (cerebellum/brainstem) of frozen brain onto flat OCT base using additional OCT compound and non-serrated tweezers.
Note
You may need to gently hold the brain in place until the OCT has frozen.

Crush dry ice and surround the OCT embedded brain, pressing gently to compact the dry ice and leaving only the most rostral aspect exposed to begin cutting (olfactory bulb/pre-frontal cortex).
Section Thikness30 µm tissue sections.

  • Ensure microtome blade and clamp are slightly wet, this helps to avoid tissue crumpling or sticking to blade.
  • Sections must be transferred quickly into wells containing anti-freeze solution using a paint brush.
  • If tissue becomes too soft or loses its visual homogeneity due to thawing (sections will start to shred) pause sectioning, top up crushed dry ice around brain sample holder, wait to re-freeze and continue.
  • To collect 12 section series', transfer sections sequentially into 12 labelled vials - section 1 is placed into vial 1, section 2 into vial 2, and so on until the last vial is reached, at which point the process begins again and the next section is placed into vial 1 again.
Once all sections have been cut for a given brain tissue, store sections in anti-freeze solution at Temperature-20 °C .