CYTOCHEMISTRY AND ELECTRON MICROSCOPY

TLDR

The authors developed a series of aldehyde fixatives (4–6.5 % glutaraldehyde, 4 % glyoxal, 12.5 % hydroxyadipaldehyde, 10 % crotonaldehyde, 5 % pyruvic aldehyde, 10 % acetaldehyde, 5 % methacrolein) prepared in 0.1–0.2 M cacodylate or phosphate buffers (pH 6.5–7.6) with sucrose, applied for 0.5–24 h fixation, stored at 4 °C, and used for enzyme histochemistry and electron microscopy, with optional 1–2 % osmium tetroxide postfixation. Fixation with these aldehydes obscured membranous differentiation and altered nuclear structure, but osmium postfixation restored images comparable to osmium alone; most enzymes (e.g., esterase, acetylcholinesterase, phosphatases, diaphorases) remained detectable, while cytochrome oxidase, succinate dehydrogenase, and glucose‑6‑phosphatase were preserved mainly after hydroxyadipaldehyde and glyoxal, and enzyme activity could be localized to fine structure.

Abstract

The aldehydes introduced in this paper and the more appropriate concentrations for their general use as fixatives are: 4 to 6.5 per cent glutaraldehyde, 4 per cent glyoxal, 12.5 per cent hydroxyadipaldehyde, 10 per cent crotonaldehyde, 5 per cent pyruvic aldehyde, 10 per cent acetaldehyde, and 5 per cent methacrolein. These were prepared as cacodylate- or phosphate-buffered solutions (0.1 to 0.2 M, pH 6.5 to 7.6) that, with the exception of glutaraldehyde, contained sucrose (0.22 to 0.55 M). After fixation of from 0.5 hour to 24 hours, the blocks were stored in cold (4°C) buffer (0.1 M) plus sucrose (0.22 M). This material was used for enzyme histochemistry, for electron microscopy (both with and without a second fixation with 1 or 2 per cent osmium tetroxide) after Epon embedding, and for the combination of the two techniques. After fixation in aldehyde, membranous differentiations of the cell were not apparent and the nuclear structure differed from that commonly observed with osmium tetroxide. A postfixation in osmium tetroxide, even after long periods of storage, developed an image that—notable in the case of glutaraldehyde—was largely indistinguishable from that of tissues fixed under optimal conditions with osmium tetroxide alone. Aliesterase, acetylcholinesterase, alkaline phosphatase, acid phosphatase, 5-nucleotidase, adenosine triphosphatase, and DPNH and TPNH diaphorase activities were demonstrable histochemically after most of the fixatives. Cytochrome oxidase, succinic dehydrogenase, and glucose-6-phosphatase were retained after hydroxyaldipaldehyde and, to a lesser extent, after glyoxal fixation. The final product of the activity of several of the above-mentioned enzymes was localized in relation to the fine structure. For this purpose the double fixation procedure was used, selecting in each case the appropriate aldehyde.