Diff of 4.4.1 Staining of cartilage and bone in whole embryos, larvae and adults by Alcian blue and Alizarin red

The added line is THIS COLOR.
The deleted line is THIS COLOR.
Go to 4.4.1 Staining of cartilage and bone in whole embryos, larvae and adults by Alcian blue and Alizarin red.
[[FrontPage]]

*4.4.1 Staining of cartilage and bone in whole embryos, larvae and adults by Alcian blue and Alizarin red [#cb26369b]

Keiji Inohaya~
Tokyo Institute of Technology

**4.4.1.1 Reagents [#t852edc6]

- Formalin
- 4% paraformaldehyde with 1% 5N sodium hydrate
- Alcian blue solution (0.1g Alcian blue, 70ml ethanol, 30ml glacial acetic acid)
- Enzyme solution I (10mg/ml trypsin in 30% saturated aqueous sodium borate)
- Enzyme solution II (0.1-1% trypsin in 30% saturated aqueous sodium borate)
- Alizarin Red solution I (2ml saturated Alizarin red S in ethanol / 8ml 0.5% KOH)
- Alizarin red solution II (0.4ml saturated Alizarin red S in ethanol / 10ml 0.5% KOH)
- Ethanol series (95%, 75%, 50%, 25%)
- 0.5% aqueous KOH 
- Hydrogen peroxide
- Glycerin

**4.4.1.2 Method [#t85f2bfd]

***Method 1: Older larvae or adults [#u66e431c]

+ Fix the specimens with 10% formalin / phospate-buffered saline (PBS) overnight or longer.
+ Wash the specimens in PBS.
+ Transfer to distilled H2O.
+ Stain for overnight in fresh Alcian blue solution at room temperature.
+ Wash in 95% ethaol for several times.
+ Transfer through 75%, 50%, and 25% ethanol.
+ Transfer to distilled H2O.
+ Digest in &lsquo;Enzyme solution I&rsquo; overnight or longer at room temperature. Remove scale with forceps. Change the enzyme solution for several times. Continue until bones and cartilage are clearly visible.
+ Transfer to 0.5% aqueous KOH.
+ Stain in &lsquo;Alizarin Red solution I&rsquo; at room temperature until bones are distinctly red.
+ Wash in 0.5% aqueous KOH for several times.
+ Transfer in graded series of 0.5% KOH / glycerine (3:1, 1:1, 1:3). 
(Option: 3 drops of 3% H2O2 are added per 100ml solution to bleach pigments of specimens.)
+ Store in 100% glycerine. A few crystals of thymol may be added to inhibit growth of molds and bacteria.

***Method 2: Alizarin red staining for embryos or young larvae [#v054d3a3]

+ Fix the specimens in 4% paraformaldehyde with 1% 5N NaOH overnight at room temperature. 
+ Wash the specimens in PBST (0.1% Tween 20 in PBS).
+ Stain in &lsquo;Alizarin red solution II&rsquo; at room temperature for several hours or overnight. 
+ Wash in 0.5% aqueous KOH for several times.
+ Transfer in graded series of glycerine (15%, 30%, 60%, 80% in 0.5% KOH). 
+ Store in 100% glycerine.

***Method 3: Alcian blue staining for embryos or young larvae [#i49d25a8]

+ Fix the specimens in 4% paraformaldehyde overnight at room temperature. 
+ Wash the specimens in PBST.
+ Stain in &lsquo;Alcian blue solution&rsquo; overnight.
+ Wash in ethanol for several times. 
+ Transfer through an ethanol series to PBST. 
+ Bleach with hydrogen peroxide (3% H2O2 in 1% KOH) for 1 hour or until eyes of specimens become transparent.
+ Wash in PBST for several times. 
+ Digest in &lsquo;Enzyme solution II&rsquo; at room temperature for less than 1 hour.
+ Wash in PBST for several times.
+ Store in PBST.

**4.4.1.3 References [#ocf4d559]

Dingerkus G, Uhler LD (1977) Enzyme clearing of alcian blue stained whole small vertebrates for demonstration of cartilage. Stain Technol. 52: 229-232.

Inohaya K, Kudo A (2000). Temporal and spatial patterns of cbfa1 expression during embryonic development in the teleost, Oryzias latipes. Dev. Genes. Evol. 210, 570-574.

Yasutake J, Inohaya K, Kudo A (2004). Twist functions in vertebral column formation in medaka, Oryzias latipes. Mech. Dev. 121, 883-894.