Sakata-Haga H et al. (2018), A rapid and nondestructive protocol for whole-m...

XB-ART-54882

Sci Rep 2018 May 10;81:7453. doi: 10.1038/s41598-018-25836-4.

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

Sakata-Haga H , Uchishiba M , Shimada H , Tsukada T , Mitani M , Arikawa T , Shoji H , Hatta T .

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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.

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	Figure 1. Efficacy of our new fixative. The medaka, zebrafish, and Xenopus after immersion in the new fixative for 24 h at 42 °C (D–F, respectively). These were decolored and cleared compared with untreated specimens (A–C, respectively). The decolorization and clearing increased with longer incubation in the fixative (G–I). After 48 h of incubation, the characteristic stripe pattern of the zebrafish disappeared (H). The zebrafish incubation in the enhancement solution for 24 h at 42 °C following immersion in the fixative for 24 h at 42 °C (J). Scale bar = 1 cm.
	Figure 2. Whole-body images of the specimens after bone staining by the new procedure. The images show the medaka, zebrafish, and Xenopus incubated with the fixative for 24 h at 42 °C (upper row; A–C, respectively), incubated with the fixative for 48 h at 42 °C (middle row; D,E, and F, respectively), and incubated with fixative for 24 h at 42 °C followed by incubation with the enhancement solution for 24 h at 42 °C (lower row; G). Scale bar = 1 cm.
	Figure 3. A flowchart of the rapid (RAP) system for bone staining (RAP-B). The new procedure for bone staining which is rapid, nondestructive, and allowed to obtain high-definition, fine bone-stained specimens, was based on our RAP system. Cartilage staining (RAP-C) can be applied as single staining or optionally added before the bone staining for double staining of bone and cartilage (RAP-B/C, Supplementary Fig. S8). FIXATIVE: 5% formalin, 5% Triton X-100, 1% potassium hydroxide (KOH); B-STAINING SOLUTION: 0.05% alizarin red S, 20% ethylene glycol, 1% KOH; C-STAINING SOLUTION: 50–70% ethanol, 20% acetate, 0.015–0.02% alcian blue; CLEARING SOLUTION: 20% Tween 20, 1% KOH; ENHANCEMENT SOLUTION: 20% ethylene glycol, 5% Triton X-100, 1% KOH.
	Figure 4. Whole-body (A) and magnification images of the tail fine ray (B), the skull (C), the vertebral column (D), and the anal fine ray (E) of the zebrafish after bone staining by our new procedure. Whole-body (F), and magnification fluorescence images the tail fine ray (G), the skull (H), the vertebral column (I), and the anal fine ray (J), and high- magnification fluorescence images of the orbit (K), the vertebral bones (L), and the joint part of proximal, intermedius, and distal pterygiophores of the anal fine ray (M) of the same zebrafish specimen shown in (A–E). The fluorescence images were reconstructed by maximum intensity projection (MIP) after image stitching. Scale bar in A and F = 5 mm; Scale bar in B, C, D, E, G, H, I, and J = 2 mm; Scale bars in K, L, and M = 0.5 mm.

References [+] :

ADKINS, ALIZARIN RED S AS AN INTRAVITAL FLUOROCHROME IN MINERALIZING TISSUES. 1965, Pubmed