Dec 19, 2023
Mouse tissue collection
- 1National Institute of Health
Protocol Citation: Tae-Un Han 2023. Mouse tissue collection. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj3ejwlk5/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: December 19, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 92515
Keywords: ASAPCRN
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000458
Abstract
This protocol is about collecting mouse tissues for immunohistochemistry or biochemistry experiment. While it is mainly used for brain tissues, it also can be used for other visceral tissues.
Perfusion
Perfusion
Mice are anesthetized with Avertin (1mL/25g body weight).
Immobilize a mouse and cut through the skin and muscles in the lower abdomen exposing the liver using a sharp scissors.
Cut through the diaphragm, then down the right and left sides of the chest, through the ribs, until the heart is fully visible.
Grasp the heart firmly near the apex with a blunt forceps, then insert the butterfly needle gently into the left ventricle.
Using a small, sharp scissors, make a small cut in the right atrium to serve as a drain.
Flow 1x PBS (Quality Biological) containing 1 IU Heparin/mL (about 00:00:30 ) followed by 4 % volume paraformaldehyde for fixation until evidence of stiffness of tissues is observed.
30s
Tissues are collected and post-fixed in 4 % volume PFA at 4°C for about 24:00:00
1d
For floating sections, tissues are transferred to 30% sucrose for cryoprotection, then kept at 4 °C for about 48:00:00 until tissues sink to the bottom of the vessel.
2d
The floating sections are collected with microtome combined with freezing unit. After getting 30~40 um thickness of floating sections, they can be stored in PBS-sodium azide (0.05 % volume ) solution for 1344:00:00 maximum.
8w
For paraffin sections for histopathology evaluation, tissues are transferred to 70 % volume ethanol after fixation then kept at 4 °C until usage.
Frozen tissues
Frozen tissues
Tissues for biochemical experiment such as gene expression analysis, protein assay or lipidomics are collected using snap-freezing in dry ice without perfusion, then stored at -80 °C o C.