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4.4.1 Staining of cartilage and bone in whole embryos, larvae and adults by Alcian blue and Alizarin red†
Keiji Inohaya
Tokyo Institute of Technology
4.4.1.1 Reagents†
- 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†
Method 1: Older larvae or adults†
- 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 ‘Enzyme solution I’ 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 ‘Alizarin Red solution I’ 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†
- 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 ‘Alizarin red solution II’ 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†
- Fix the specimens in 4% paraformaldehyde overnight at room temperature.
- Wash the specimens in PBST.
- Stain in ‘Alcian blue solution’ 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 ‘Enzyme solution II’ at room temperature for less than 1 hour.
- Wash in PBST for several times.
- Store in PBST.
4.4.1.3 References†
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.