Trans-cardiac perfusion of neonatal mice and immunofluorescence of the whole body as a method to study nervous system development

• Published in: PloS one Vol. 17; no. 10; p. e0275780
• Main Authors: Pérez Arévalo, Andrea, Lutz, Anne-Kathrin, Atanasova, Ekaterina, Boeckers, Tobias M.
• Format: Journal Article
• Language: English
• Published: San Francisco Public Library of Science 13.10.2022; Public Library of Science (PLoS)
• Subjects: Analysis; Animals; Biology and Life Sciences; Brain; Cellular structure; Circulatory system; Developmental stages; Fixation; Fluorescent antibody technique; Health aspects; Heart; Immunofluorescence; Immunohistochemistry; Infants (Newborn); Juveniles; Lab Protocol; Medical research; Medicine and Health Sciences; Methods; Neonates; Nervous system; Newborn babies; Perfusion; Physical Sciences; Research and analysis methods; Rodents; Skin; Spinal cord; Staining; Structural analysis; Tissue analysis; Tissues; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0275780

Whole animal perfusion is a well-established method that has been used for the past decades in multiple research fields. Particularly, it has been very important for the study of the brain. The rapid and uniform fixation of tissue is essential for the preservation of its integrity and the study of complex structures. For small tissue pieces submerging in formaldehyde solution oftentimes is sufficient to get a good fixation, larger tissues or organs with a more complicated structure present a greater difficulty. Here, we report the precise parameters to successfully perform trans-cardiac perfusion of neonatal mouse pups that allows a uniform fixation of the whole body for subsequent structural analysis and immunohistochemistry. In comparison to standard perfusion procedures of adult mice, changes in the pump velocity, the buffer volume and in the needle size lead to high quality fixation of neonatal mice pups. Further, we present a whole-body section staining, which results in a highly specific immunofluorescence signal suited for detailed analysis of multiple tissues or systems at the same time. Thus, our protocol provides a reproducible and reliable method for neonatal perfusion and staining that can rapidly be applied in any laboratory. It allows a high quality analysis of cellular structures and expression profiles at early developmental stages.