Antigenicity is preserved with fixative solutions used in human gross anatomy: A mice brain immunohistochemistry study

Abstract

BackgroundHistology remains the gold-standard to assess human brain biology, so ex vivo studies using tissue from brain banks are standard practice in neuroscientific research. However, a larger number of specimens could be obtained from gross anatomy laboratories. These specimens are fixed with solutions appropriate for dissections, but whether they also preserve brain tissue antigenicity is unclear. Therefore, we perfused mice brains with solutions used for human body preservation to assess and compare the tissue quality and antigenicity of the main cell populations.Materials and methodsTwenty-eight C57BL/6J mice were perfused with 4% formaldehyde (FAS, N = 9), salt-saturated solution (SSS, N = 9), and alcohol solution (AS, N = 10). The brains were cut into 40 μm sections for antigenicity analysis and were assessed by immunohistochemistry of four antigens: neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP astrocytes), ionized calcium-binding adaptor molecule 1 (Iba1-microglia), and myelin proteolipid protein (PLP). We compared the fixatives according to multiple variables: perfusion quality, ease of manipulation, tissue quality, immunohistochemistry quality, and antigenicity preservation.ResultsThe perfusion quality was better using FAS and worse using AS. The manipulation was very poor in SSS brains. FAS- and AS-fixed brains showed higher tissue and immunohistochemistry quality than the SSS brains. All antigens were readily observed in every specimen, regardless of the fixative solution.ConclusionSolutions designed to preserve specimens for human gross anatomy dissections also preserve tissue antigenicity in different brain cells. This offers opportunities for the use of human brains fixed in gross anatomy laboratories to assess normal or pathological conditions.

Comparison of histological procedures and antigenicity of human post-mortem brains fixed with solutions used in gross anatomy laboratories

“…Therefore, sufficient quality IHC procedures are possible following the application of an antigen retrieval protocol. These observations differ from what we reported in our previous study in mice, in which the antigenicity of the four main cell populations was preserved and homogeneously distributed across all the types of fixation (Frigon & al., 2022), even when no antigen retrieval protocol was applied prior to the IHC procedures. This also supports the differences in mice and human brain tissue preservation properties.…”

“…The human tissue samples were relatively stiff and well-fixed macroscopically, and we found mostly that sections from AFS fixed specimens were easy to manipulate, and slightly more difficult in cases fixed with SSS. Nevertheless, SSS-fixed human sections were relatively easier to manipulate (stiffer) than the mouse sections of brains fixed with the same solutions (Frigon & al., 2022). This could be related to their different capacity to absorb the fixative (diffusion), related to their differences in shape, size, gyrification, and vascularization circuits across species (Ventura-Antunes et al, 2013).…”

“…Third, free-floating tissue sections stained with IHC procedures might show heterogeneous labeling, since the staining depends on the level of antibody penetration and the tissue background. Analyzes might therefore be affected by the protocol itself, which was also evaluated as a variable of interest in our previous study in mice brains (Frigon & al., 2022). Therefore, the protocols were already tested and validated for this study, so we excluded the antibody penetration and tissue background quality as variables here.…”

Ex vivo, in situ perfusion protocol for human brain fixation compatible with microscopy, MRI techniques, and anatomical studies

“…Thus, anatomy departments are in a privileged position to overcome limitations associated with the restricted availability of brain tissue of brain banks, since is possible to fix brains via the same process used to embalm human bodies. Moreover, a recent report by Frigon et al ( 2022 ) shows that mice perfused with several different fixatives used in embalming bodies retained antigenicity, allowing immunoreactivity to several antibodies to be demonstrated.…”

“…Fixation of the donated body through the vascular system, either by the carotid or the femoral arteries, is the standard procedure in most anatomy departments. However, the solutions used for body fixation often preclude histological processing of the brain (see Frigon et al, 2022 ). Despite anatomy departments receiving numerous body donations, most of these bodies are not fully exploited when it comes to human brain studies.…”