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

Abstract

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.

Administration of adipose-derived stem cells extracellular vesicles in a murine model of spinal muscular atrophy: effects of a new potential therapeutic strategy

“…For the histological analysis of brain ( n = 2 WT), spinal cord ( n = 5 SMA-EVs; 6 SMA-PBS; 3 WT), gastrocnemius and quadriceps muscles ( n = 5 SMA-EVs; 7 SMA-PBS; 6 WT), at P10 animals were deeply anesthetized by gaseous anaesthesia (isoflurane 5%). After confirming that the animals had reached a state of deep anesthesia, pups were transcardically perfused with phosphate buffer 0.1 M (PB, pH 7.4), followed by paraformaldehyde (PFA) 4%, as described in [ 51 ] and reported in [ 52 ]. The tissues were dissected out and post-fixed in PFA 4% overnight at 4 °C.…”

The adhesion GPCR ADGRV1 controls glutamate homeostasis in hippocampal astrocytes supporting neuron development: first insights into to pathophysiology ofADGRV1-associated epilepsy

“…16 µm thick cryosections and placed onto Poly-L-Lysine (Sigma-Aldrich, P4832-50ML) coated coverslips. After permeabilization in 0.2% Triton X-100 for 10 min (Sigma-Aldrich, 102533092) in PBS, immunostaining performed as previously described (Arévalo et al, 2022). Fluorescence intensity immunoreactivity in mouse hippocampi was analyzed using a build-in ImageJ/Fiji plugin (https://fiji.sc/).…”

Importancia de la técnica de perfusión intracardiaca para los estudios in vivo en Odontología

“…La principal ventaja consiste en la perfusión directa del fijador a través del sistema circulatorio, por lo que puede alcanzar cada tejido del cuerpo utilizando la red vascular natural 3 , 4 . En este método, el líquido fijador se introduce en el corazón, concretamente en el ventrículo izquierdo, y es distribuido a través del sistema arterial a los órganos diana 2 . El conocimiento de la fisiología del sistema circulatorio del animal de experimentación, así como de su anatomía, es muy importante para que el investigador lleve a cabo esta técnica de manera exitosa.…”

“…Si bien es cierto que otro método, denominado fijación por inmersión, es bastante utilizado debido a su practicidad, rapidez y a que no requiere un proceso de adiestramiento tan estricto como el de la perfusión intracardiaca, sus resultados pueden verse afectados por muchos factores, como el tamaño de la muestra, el tipo de tejido, la concentración de la solución fijadora, etc. Por ello, existe la posibilidad de que el líquido fijador no llegue a las capas celulares internas o que lo haga, pero no de manera uniforme 2 , 8 .…”

“…El largo proceso desde la obtención de las muestras hasta su tinción final implica una serie de protocolos en cada etapa que aseguren una adecuada imagen para el análisis histomorfométrico. Aunque cada paso es sumamente importante, estudiar la estructura y composición de los tejidos de la manera más confiable requiere la preservación óptima de estos 2 , 3 . Tal preservación se da principalmente a través de la denominada “fijación” de tejidos.…”