Glutaraldehyde: A review of its fixative effects on nucleic acids, proteins, lipids, and carbohydrates

Abstract

Tissue banking methods such as brain banking often face a trade-off between morphological and molecular preservation. For example, cryopreservation is preferred for subsequent molecular assays, while fixation by aldehyde crosslinking is commonly used for microscopy. Among aldehyde fixatives, formaldehyde is often considered better for immunohistochemistry, while glutaraldehyde is often considered better for electron microscopy. However, it is unclear whether morphological versus molecular preservation trade-offs reflect fundamental biology or technology limitations. As a window into this discussion, in this narrative review, I evaluate the literature regarding the effects of glutaraldehyde on molecular preservation, with an emphasis on nervous system tissue. Available evidence suggests that crosslinking with glutaraldehyde has minimal direct effects on most molecular features, with a few critical exceptions such as protein conformation. On the other hand, glutaraldehyde fixation frequently fails to retain non-directly crosslinked molecules such as lipid and carbohydrate species during subsequent dehydration steps. Further, as a result of probe diffusion limitations or strong covalent interactions with glutaraldehyde, many molecules can be more difficult to visualize or otherwise measure in tissue fixed with glutaraldehyde. As a practical guide for investigators that are considering using glutaraldehyde, I also point out representative molecular assays that have been performed in tissue fixed with glutaraldehyde, and how this set of assays could be improved and expanded in the future.

Intranuclear HSV-1 DNA ejection induces major mechanical transformations suggesting mechanoprotection of nucleus integrity

“…After incubation, the sample was fixated with 2% GA, washed, and resuspended in capsid-binding buffer (CBB), after which it was deposited on hydrophobically modified AFM slides for imaging ( Materials and Methods ). Fixation of the nuclei in GA cross-links the lamina meshwork ( 39 , 40 ), which increases nucleus surface stiffness by ∼10 times (as shown below). Since fixation provides increased nuclear surface rigidity, it significantly improves the imaging resolution of capsids on the nuclear surface.…”

“…Thus, by collecting AFM force volume maps on the nuclear surface without and with HSV-1 capsids docked at NPCs, we reveal the mechanical response of the nuclear chromatin. Separately, we also determine the nuclear lamina’s mechanical response to AFM indentation by decoupling the mechanical response of chromatin stiffness from nuclear lamina mechanics through chemical cross-linking of the lamina meshwork with GA, which does not cross-link nucleic acids ( 39 , 40 ). Thus, GA fixation increases lamina shell rigidity above that of chromatin rigidity, making the contribution of chromatin stiffness to the overall nucleus mechanics negligible (this approach is explained in more detail in Mechanical Response of the Nuclear Lamina to HSV-1 DNA Ejection ).…”

“…Thus, to measure and separate the nuclear lamina mechanical response to AFM force volume mapping from the contribution from chromatin to the overall nucleus stiffness, nuclear lamina rigidity needs to be increased above that of chromatin. This can be achieved by chemically cross-linking the nuclear lamina shell with 2% GA. GA is a cross-linking agent that mainly cross-links proteins but not nucleic acids ( 39 ), freely crosses nuclear membranes, does not react with either DNA or RNA at room temperature (therefore, GA fixation preserves chromatin structure without inducing major artifacts; Ref. 39 ), and has been shown to effectively cross-link lamina structure ( 40 ).…”

“…This can be achieved by chemically cross-linking the nuclear lamina shell with 2% GA. GA is a cross-linking agent that mainly cross-links proteins but not nucleic acids ( 39 ), freely crosses nuclear membranes, does not react with either DNA or RNA at room temperature (therefore, GA fixation preserves chromatin structure without inducing major artifacts; Ref. 39 ), and has been shown to effectively cross-link lamina structure ( 40 ). Electron microscopic localization of lamins in isolated rat liver nuclei showed that lamins are present at the nuclear periphery and are absent from more internal regions of the nucleus ( 40 ).…”

3D molecular phenotyping of cleared human brain tissues with light-sheet fluorescence microscopy

“…However, we encountered several hurdles for performing volumetric reconstruction of the human cortex. The first challenge is the long fixation time, which could cause a variety of changes in the 3D structure of proteins, decreasing the possibility to detect specific epitopes for phenotyping and connectomes analyses (42,43). Also, the presence of lipopigments -such as lipofuscin and neuromelanin (44)(45)(46) -and blood, have remained an insuperable hurdle during the acquisition process, causing spurious signals that make cell identification and counting more complex.…”