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

Scientific Reports
Hiromi Sakata-HagaToshihisa Hatta

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.

Citations

Aug 28, 2020·Frontiers in Endocrinology·Francesca TonelliAntonella Forlino
Oct 8, 2020·The Anatomical Record : Advances in Integrative Anatomy and Evolutionary Biology·Nicholas Zinck, Tamara A Franz-Odendaal
Jan 1, 2021·Human Mutation·Elizabeth A TerhuneNancy Hadley Miller
Jan 24, 2021·Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research·Kristin DietrichFranziska Knopf
May 16, 2021·American Journal of Human Genetics·Lore PottieBert Callewaert
May 7, 2021·Journal of Bone and Mineral Research : the Official Journal of the American Society for Bone and Mineral Research·Jan Willem BekPaul J Coucke
Jul 21, 2021·Child's Nervous System : ChNS : Official Journal of the International Society for Pediatric Neurosurgery·Takuya AkaiSatoshi Kuroda
May 10, 2019·ACS Nano·Weinan Xu, David H Gracias
Nov 9, 2021·Frontiers in Cell and Developmental Biology·Carlos Camacho-MacorraMarcos J Cardozo

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Methods Mentioned

BETA
RAP
confocal microscopy

Software Mentioned

Zen 2013

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