Dec 19, 2024

Public workspaceIntracardiac perfusion with buffer for anatomical studies

 Forked from Intracardiac perfusion with fixative for anatomical studies
DOI
dx.doi.org/10.17504/protocols.io.3byl4kpp8vo5/v1
  • 1University of Melbourne
  • SPARC
    Tech. support email: info@neuinfo.org
Nicole Wiedmann
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DOI: dx.doi.org/10.17504/protocols.io.3byl4kpp8vo5/v1
Protocol Citation: Janet R Keast, Peregrine B Osborne, Nicole Wiedmann 2024. Intracardiac perfusion with buffer for anatomical studies. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4kpp8vo5/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
Created: November 01, 2021
Last Modified: December 19, 2024
Protocol Integer ID: 54689
Keywords: tissue preservation, tissue fixation
Funders Acknowledgements:
NIH SPARC
Grant ID: 3OT2OD023872
Abstract
This protocol is suitable for preserving tissues for anatomical studies of organs and major pelvic ganglia 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 fixative: 4% paraformaldehyde in phosphate buffered saline (PBS)Contributed by users

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.

Fixative: 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4. This is made up no longer than 48h prior to use, stored at 4C and brought to room temperature on the day of 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).
Dissect tissues required for analysis and place in fixative overnight at 4 degrees.
Post-perfusion
Post-perfusion
Wash tissues with phosphate-buffered saline (PBS; 0.1 M, pH7.2), 3 x 30 min washes.
Store in PBS containing 0.1% sodium azide at 4C until used for immunohistochemistry or other microscopic analysis.