A rapid and nondestructive protocol for whole-mount bone staining of small fish and Xenopus

Tomohiro Arikawa,Hiroki Shoji,Toshihisa Hatta,Mayumi Mitani,Maimi Uchishiba,Tsuyoshi Tsukada,Hiromi Sakata-Haga,Hiroki Shimada

doi:10.1038/s41598-018-25836-4

Tomohiro Arikawa, Hiroki Shoji + Show 6 more

Open Access

https://doi.org/10.1038/s41598-018-25836-4

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Abstract

Here we propose a new protocol for whole-mount bone staining, which allows the rapid preparation of highly cleared and nondestructive specimens. It only takes 3 days to complete whole procedure for small vertebrates, such as medaka, zebrafish, and Xenopus frogs. In this procedure, we used a newly developed fixative containing formalin, Triton X-100, and potassium hydroxide, which allows the fixation, decolorization, and transparentization of specimens at the same time. A bone staining solution containing alizarin red S with ethylene glycol and a clearing solution containing Tween 20 and potassium hydroxide also contributed the specificity and swiftness of this new system. As expected, although details of the skeletal system could be observed in specimens with high transparency, it was noteworthy that high-resolution fluorescence images acquired using zoom microscopes clearly delineated the shape of each bone. This new procedure would be expected to be widely used as a standard procedure for bone staining in the testing the developmental toxicity of chemicals and in the screening test of knockout or mutant animals.

Highlights

We propose a new protocol for whole-mount bone staining, which allows the rapid preparation of highly cleared and nondestructive specimens
Staining bones with alizarin red S after clearing away the soft tissue with a solution containing potassium hydroxide (KOH) was widely used for many years as a standard method for bone staining in small vertebrates[2]
The Scale reagent, a urea-based agent used to make soft tissue transparent[8], has been used following bone staining with alizarin red S9; it can be difficult to use this solution for tests that require large-scale analysis, such as reproductive toxicology testing

Summary

Introduction

We propose a new protocol for whole-mount bone staining, which allows the rapid preparation of highly cleared and nondestructive specimens It only takes 3 days to complete whole procedure for small vertebrates, such as medaka, zebrafish, and Xenopus frogs. Staining bones with alizarin red S after clearing away the soft tissue with a solution containing potassium hydroxide (KOH) was widely used for many years as a standard method for bone staining in small vertebrates[2]. A bone staining procedure that clears the specimen without any artificial destruction would provide a valuable tool, which would allow the establishment of a nondestructive bone staining protocol that retains small or incompletely calcified immature bones in their intact position at a microscopic level. The procedure is rapid and relatively simple, and could be used to stain bones for a large number of specimens at once with no tissue destruction

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