A standardized technique for the in toto epoxy resin embedding and precipitate‐free staining of small specimens covered by strong protective outer surfaces

Abstract

SUMMARY A method is described for the in toto embedding and precipitate‐free staining of small invertebrates covered by strong protective outer surfaces. The difficulties of tissue infiltration in undissected animals with cuticles were overcome by the use of ethanol and 1,4‐dioxane as dehydrating agents, and Spurr's medium as the embedding resin. (1) Fixation as appropriate, followed by embedment in aqueous agar; (2) dehydration: 50% ethanol, 70% ethanol, 2 times 100% 1,4‐dioxane, 15–20 min for each step; (3) infiltration: dioxane:resin 1:1 90 min, dioxane:resin 1:3 90 min, pure resin overnight, changed once for an additional 10 h, all steps with constant shaking; (4) polymerization 16 h at 343 K; (5) sectioning; (6) staining: 0·9% cacodylate buffered KMnO4 (pH 6·5) 30–60 s, washing, Reynolds' lead citrate 5 min, washing by grid floating. The advantage of the method is discussed in relation to serial sectioning of entire invertebrates which could not be dissected prior to embedding.

Ultrastructure, protein synthesis and secretion of day-6 rabbit blastocysts cultured in a chemically defined, protein-free medium

“…Ultrathin sections (50 nm) were prepared on a Sorvall MT 6000 ultramicrotome. Sections were double-stained with 0.9% KMnO4 (Mothes-Wagner et al 1984) and lead citrate (Reynolds 1963), and were examined with a Hitachi HU-12 ST electron microscope at 60 kV.…”

Post-embryonic development of the two-spotted spider miteTetranychus urticaewith special reference to the differentiation of the female reproductive system

“…Semithin sections were cut with glass knives on an LKB pyramitome and stained with alcoholic methylene blue [22]. Ultrathin sections were cut with a diamond knife on an LKB ultratome and stained with KMn0 4 and lead citrate [22]. Sections were observed with a Leitz diavert microscope with standard interference equipment or a Hitachi HU 12 A electron microscope, respectively.…”

“…After dehydration in graded ethanol solutions and 1,4-dioxane [22], polymerization was effected in Spurr's medium [22]. Semithin sections were cut with glass knives on an LKB pyramitome and stained with alcoholic methylene blue [22]. Ultrathin sections were cut with a diamond knife on an LKB ultratome and stained with KMn0 4 and lead citrate [22].…”

“…For light and electron microscopy, animals of a mixed population were fixed in 4% collidine-buffered glutaraldehyde for 2 days followed by osmication (1% buffered 205 Os0 4 , pH 7.4) for 15 min at room temperature. After dehydration in graded ethanol solutions and 1,4-dioxane [22], polymerization was effected in Spurr's medium [22]. Semithin sections were cut with glass knives on an LKB pyramitome and stained with alcoholic methylene blue [22].…”