Dec 14, 2021

Public workspaceIntracardiac perfusion with fixative for ultrastructural neuroanatomical studies

Forked from a private protocol
DOI
dx.doi.org/10.17504/protocols.io.b2ssqeee
  • 1University of Melbourne
  • SPARC
    Tech. support email: info@neuinfo.org
Janet R Keast
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DOI: dx.doi.org/10.17504/protocols.io.b2ssqeee
Protocol Citation: Janet R Keast, Peregrine Osborne 2021. Intracardiac perfusion with fixative for ultrastructural neuroanatomical studies. protocols.io https://dx.doi.org/10.17504/protocols.io.b2ssqeee
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 12, 2021
Last Modified: November 27, 2023
Protocol Integer ID: 55858
Keywords: tissue preservation, tissue fixation, electron microscopy, ultrastructure
Funders Acknowledgements:
NIH-SPARC
Grant ID: OT2OD023872
Abstract
This protocol is suitable for preserving tissues for ultrastructural neuroanatomical studies of peripheral nerves, ganglia, spinal cord or brain in adult rats. The protocol is performed under anesthesia and should incorporate all local requirements for standards of animal experimentation.
Materials
MATERIALS
ReagentKetamineLyppardCatalog #KETAI1
ReagentXylazineLyppardCatalog #L10605
ReagentHeparin sodium Ellar Laboratories
ReagentParaformaldehyde (prilled 95%)Sigma AldrichCatalog #441244
ReagentGlutaraldehyde (25% aqueous solution EM grade)ProSciTechCatalog #C001

Preparation for perfusion
Preparation for perfusion
Make up the following solutions:

Perfusion prewash solution: To 300 ml 0.9% sodium chloride (w/v) add 3.75 ml 1% sodium nitrite (w/v) and 0.11 ml heparin (5000 IU/ml). This is made up immediately prior to use.

Perfusion fixative: 2% paraformaldehyde and 1.25% glutaraldehyde in 0.1M phosphate buffer, pH 7.4. The buffered paraformaldehyde solution is made up no longer than 48h prior to use, stored at 4C and brought to room temperature on the day of perfusion. Glutaraldehyde is added to this solution on the day of the perfusion.
Perfusion
Perfusion
Induce anesthesia by an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg).
After opening the chest cavity, inject the left ventricle with mixture of 0.25 ml heparin (5000 IU/ml) and 0.5 ml 1% sodium nitrite.
A needle connected to tubing (T-connector to prewash and perfusion solutions), and a peristaltic pump (setting: 50 ml/min) is then inserted into the left ventricle and clamped into place using hemostats. Make a small incision in the right atrium to drain blood and perfusate during the procedure.
Perfuse with pre-wash solution until the fluid flowing from the right atrium is clear, the liver and extremities are pale (typically 2-3 min).
Perfuse with fixative for 15-20 minutes, by which time the organs have stiffened and the neural tissues will be well preserved.
Dissect tissues required for analysis and place in fixative for a further 18-24h at 4C.
Post-perfusion
Post-perfusion
Wash tissues with phosphate-buffered saline (PBS; 0.1 M, pH7.2), 3 x 30 min washes.
Store in PBS at 4C until processed further for embedding and electron microscopy.