Acetonitrile as a substitute for ethanol/propylene oxide in tissue processing for transmission electron microscopy: Comparison of fine structure and lipid solubility in mouse liver, kidney, and intestine

Abstract

Tissue processing for transmission electron microscopy (TEM) is commonly accomplished using ethanol (EtOH) as a dehydrating solvent and propylene oxide (PO) as a transition fluid. Both solvents have some undesirable properties: EtOH solubilizes lipids; PO is highly flammable, volatile, toxic, and potentially carcinogenic. Their replacement by a compound devoid of these characteristics is therefore desirable. Acetonitrile (AN) appears to be such a solvent. It is freely miscible with water, alcohols, acetone, and epoxy resins; it does not interfere with epoxy polymerization; and the resulting cured resins have excellent cutting quality and beam stability. AN is also an excellent dehydrating agent whose use does not necessitate modification of current techniques. Most importantly, the low solubility of phospholipids (PL) in AN limits the loss of membrane lipids and, hence, leads to a better preservation of tissue features.

Changes in white matter in mice resulting from low-frequency brain stimulation

“…Samples were washed twice with ultrafiltered water and allowed to equilibrate to room temperature. The samples were infiltrated and embedded in graded acetonitrile (50,70,90, and 100%) at room temperature according to the schedule of Edwards et al (57). Samples were infiltrated with EMbed-812 resin (EMS) mixed 1:1 with acetonitrile for 1 h, followed by 1:1 EMbed-812:acetonitrile for 18 h. The samples were next placed into EMbed-812 for 3 × 1 h, then placed into silicone-embedding molds filled with fresh resin, which were then baked in a 60°C oven for 48 h. Sections of 75-and 100-nm thickness were cut on a Reichert-Jung Ultracut E (Reichert-Jung) using a glass or an Ultra Diamond knife (EMS) and mounted on Formvar/ carbon-coated slot grids (EMS) or 300-mesh Cu grids (EMS).…”

Delayed Irradiation Effects on Nasal Epithelium in Patients with Nasopharyngeal Carcinoma; An Ultrastructural Study

“…The samples were then dehydrated in a series of increasing concentrations of acetonitrile and embedded in Epon. 6 Thick sections of 1 μπι were made and stained with toluidine blue, and proper areas of sectioning were selected. Thin sections (70 to 90 nm) of the selected area were grid-stained with lead cit- rate.…”

Novel procedure for high‐fidelity tendon histology

“…We used acetonitrile (Fisher Scientific), a gentle, low-viscosity solvent (0.38 cP at 58C), to complete the dehydration process. This reagent, which is also used in electron microscopy processing, does not solubilize charged phospholipids 5 and is freely miscible with EGME. Because of its low viscosity, acetonitrile readily penetrates dense specimens and ensures thorough dehydration.…”