Perfusion with 10% neutral-buffered formalin is equivalent to 4% paraformaldehyde for histopathology and immunohistochemistry in a mouse model of experimental autoimmune encephalomyelitis

Abstract

Intravascular (IV) perfusion of tissue fixative is commonly used in the field of neuroscience as the central nervous system tissues are exquisitely sensitive to handling and fixation artifacts which can affect downstream microscopic analysis. Both 10% neutral-buffered formalin (NBF) and 4% paraformaldehyde (PFA) are used, although IV perfusion with PFA is most commonly referenced. The study objective was to compare the severity of handling and fixation artifacts, semiquantitative scores of inflammatory and neurodegenerative changes, and quantitative immunohistochemistry following terminal IV perfusion of mice with either 10% NBF or 4% PFA in a model of experimental autoimmune encephalitis (EAE). The study included 24 mice; 12 were control animals not immunized and an additional 12 were immunized with PLP139–151 subcutaneously, harvested at day 20, and fixed in the same fashion. Equal numbers (4 per group) were perfused with 10% NBF or 4% PFA, and 4 were immersion-fixed in 10% NBF. NBF-perfused mice had less severe dark neuron artifact than PFA-perfused mice ( P < .001). Immersion-fixed animals had significantly higher scores for oligodendrocyte halos, dark neuron artifact, and perivascular clefts than perfusion-fixed animals. Histopathology scores in EAE mice for inflammation, demyelination, and necrosis did not differ among fixation methods. Also, no significant differences in quantitative immunohistochemistry for CD3 and Iba-1 were observed in immunized animals regardless of the method of fixation. These findings indicate that IV perfusion of mice with 10% NBF and 4% PFA are similar and adequate fixation techniques in this model.

Spontaneous early-onset neurodegeneration in the brainstem and spinal cord of NSG, NOG, and NXG mice

“… 28 , 34 , 39 , 42 This is particularly important in the case of the neuropathological changes described in our work as subtle lesions with minimal vacuolation can be easily misinterpreted as an artifact commonly seen in the histological preparations of the mouse CNS. 37 Therefore, knowledge of the full spectrum of expected neuropathological changes is crucial to inform the need for more sensitive and specific approaches ruling out potential artifacts. 35 In this context, we have been able to confirm the development of neurodegenerative changes via the identification of gliosis, neuronal degeneration/death, and immune/inflammatory cell infiltrate.…”

A histological method for marine invertebrates

“…If the samples are more than 5 cm in diameter, they should be trimmed to a smaller size to reduce tissue degeneration. This is a general histological tissue fixative that is ideally suited for cross-linking certain proteins and preventing degradation by inactivating enzymes, suggesting a standard recommended solution for formalin-fixed paraffin-embedded samples (Copper et al, 2018;Snyder et al, 2022).…”

“…Following fixation and immersion in 70% ETOH, decalcification using 7% hydrochloric acid (HCl), 5% nitric acid, and 10% ethylenediaminetetraacetic acid (EDTA) is widely used (Copper et al, 2018;Snyder et al, 2022). However, we recommend that the surgipath decalcifier solution (Surgipath Decalcifier I: SDI) be utilized to decalcify the samples' exoskeleton, mineralized tissue, and thickness-gut diets prior to routine sectioning of paraffin-embedded blocks.…”

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

“…Aun cuando es cierto que la fijación por inmersión requiere la disección previa de las muestras y su inmersión en el fijador, el tiempo en el que deben permanecer en este varía de 24 a 48 horas según el tamaño y cantidad de las muestras 2 . La fijación por perfusión intracardiaca, por su parte, requiere el tiempo en el que 50 ml de fijador (cantidad aproximada para un roedor de 3 semanas) fluyen a través de la red vascular del animal y la inmersión posfijación no debe ser mayor a 24 horas 9 , 10 .…”