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.
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