Aldehyde fixatives: Quantification of acid‐producing reactions

Abstract

Under conditions commonly used for the preservation of tissue for electron microscopy, substantial amounts of hydrogen ions were produced when either formaldehyde or glutaraldehyde was added to solutions of amines or proteins, or to tissue homogenates. In glycine-aldehyde reactions, hydrogen ions were generated in linear proportion to the glycine concentration over the range of 0.001 M to 0.100 M glycine. In the presence of 1% formaldehyde or 1% glutaraldehyde, 0.3-0.9 equivalents of acid were produced per mole of primary amine. This variation is a function of the specific primary amine used. Acid production increased with increasing formaldehyde concentration but did not iiicrease appreciably with increasing glutaraldehyde concentration. The concentration of amines in tissue is high. These amines are probably responsible for much of the acid generated when aldehydes react with tissue homogenates.In amine-aldehyde reactions, the positive charge originally associated with the parent amine is simultaneously neutralized with the formation of hydrogen ions. Thus, in tissues exposed to aldehydes, there is a decrease in the overall positive charge which is related to the amount of acid generated. The inherent buffering capacity of liver and muscle was not sufficient to prevent large pH decreases when aldehydes were added to homogenates of these tissues. These data suggest that significant but transient pH decreases may occur within cells exposed to aldehydes. To minimize the aldehyde-induced pH decreases, a buf'fer should be chosen so that its pK, is 0.2-0.3 pH units less than the pH desired for a particular experiment. In addition, buffer concentrations of at least 0.10 M are suggested for fixatives in order to provide adequate buffering capacity during tissue preservation.

Unconventional antigen retrieval for carbohydrate and protein antigens

“…Hence it is unclear if exposure of ultrathin cryosections to an acidic milieu reverses such crosslinks and to what extent, if at all. It has been shown that the ratio of the glutaraldehyde concentration to the amine concentration determines various aspects of the fixation process, e.g., the nature of the products formed (Johnson 1985). On this point, the glutaraldehyde concentration of 0.1% in our fixative was rather low.…”

Mitochondrial translocation of α-synuclein is promoted by intracellular acidification

“…The released protons will be partially neutralized by the inherent buffering capacity of cytoplasmic components, although this capacity is relatively low [31]. No effect of formaldehyde on pH was observed during the in vitro mitochondrial binding reactions.…”

“…It is known, but generally unappreciated that fixation with aldehydes causes the release of protons, primarily derived from the reaction with primary amines [31,32]. The released protons will be partially neutralized by the inherent buffering capacity of cytoplasmic components, although this capacity is relatively low [31].…”

Ultrarapid freezing reveals that skeletal muscle caveolae are semipermanent structures

“…(This may mean that surface fibers are exposed to high concentrations of glutaraldehyde more quickly than are the fibers in perfusion-fixed muscle, where diffusion from the restricted volume of the capillary bed may delay fixation for several additional seconds and expose the fiber to slowly increasing concentration of fixative.) There also appear to be other aspects of fixation chemistry influencing these reported changes (i.e., hypoxia, acid production, and/or loss of positive charges on membranes (Johnson, 1985(Johnson, , 1986. Moreover, the current and previous data (Lee et al, 1986) raise additional concerns (Chandler and Heuser, 1979) as to the extent different chemical fixation methods induce artifactual changes in the morphology of other (membranous and cytoplasmic) structures.…”