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<br />distilled water; mix: well and allow <br />excess sodium borate to settle; use <br />clear supernatant solution. CS, CL <br /> <br />1 % Potassium hydroxide (KOH) solution <br />In distilled water. BL,CL,BS <br /> <br />2% Potassium hydroxide solution <br />In distilled water. <br />If KOH clearing used used. CL <br /> <br />Bleaching solution <br />15% of 3% hydrogen peroxide solu- <br />tion in 1% KOH solution. BL <br /> <br />Trypsin solution <br />About 0.1-0.2 g (depending on <br />strength or activity level of trypsin) <br />per 100 ml of 30% saturated sodium <br />borate solution in distilled water; mix <br />well but do not allow to froth <br />(Taylor and Van Dyke 1985). Make <br />a fresh solution for each use; it does <br />not keep well. <br />If enzyme clearing used. CL <br /> <br />Alizarin red stain solution <br />Dissolve enough alizarin red powder <br />in 1% KOH to turn the solution <br />deep purple (about 0.1 g per 100 <br />ml). Or mix: about 1 ml of a satur- <br />ated alizarin red solution per 100 ml <br />of 1% KOH (saturated alizarin red <br />solution is prepared by dissolving <br />excess alizarin red powder in small <br />amount of distilled water, about 1.5- <br />2.0 g per 20 ml). Alizarin red stain <br />solution will keep at least one <br />week. BS <br /> <br />40% glycerin solution <br />In 1% KOH (preferred) or distilled <br />water. <br />If glycerin is used. CL <br /> <br />70% glycerin solution <br />In 1% KOH (preferred) or distilled <br />water. <br />If glycerin is used. CL <br /> <br />Fixation and Preservation: <br /> <br />1. Kill and fIx specimens in 10% buffered <br />formalin for 24-48 hours. <br /> <br />2. If specimens are to be stored more than <br />a couple days before clearing and stain- <br />ing, preserve them in 3-5% buffered for- <br />malin or alcohol (preferably via a graded <br /> <br />series of concentrations, e.g., 50% eth- <br />anol for 6-24 hours then 75% ethanol). <br />Do not soak in water between fIxative <br />and preservative solutions. <br /> <br />Cartilage Staining Procedure: <br /> <br />3. Dehydrate formalin-fIxed and preserved <br />specimens in 50% ethanol solution for 6- <br />24 hours, then in 100% or absolute eth- <br />anol for 12-24 hours. Replace the abso- <br />lute ethanol and leave at least another <br />12-24 hours. A more gradual series of <br />alcohol concentrations can be used, e.g., <br />50%, 75%, and 100% but is usually un- <br />necessary. If specimens were preserved <br />in alcohol, skip the 50% ethanol step. <br />For embryos and larvae, dehydration is <br />essential to assure minimal loss of bone <br />while in the acid stain for cartilage. <br /> <br />4. Stain specimens in alcian blue stain solu- <br />tion for 6-24 hours, no longer than nec- <br />essary to adequately stain all cartilage. <br /> <br />5. Rinse specimens in saturated sodium <br />borate solution then soak in fresh satur- <br />ated sodium borate solution for 6-24 <br />hours to neutralize (change body fluid <br />pH from acid to alkaline). <br /> <br />Bleaching: (Optional) <br /> <br />6. If specimens are heavily pigmented (such <br />that pigments would obscure desired <br />structures), bleach specimens by placing <br />them in bleaching solution and exposing <br />them to strong light until chromatophore <br />pigment is notably faded, about 20 min- <br />utes to a few hours. <br /> <br />Initial Clearing: <br /> <br />7. Enzyme method. If specimens were not <br />processed for cartilage staining, soak <br />them in saturated sodium borate solution <br />for 2-12 hours to remove remaining for- <br />malin or alcohol and adjust body fluids <br />to well above pH 7. Soak specimens in <br />trypsin solution until 75-90% of the <br />muscle tissue is cleared, typically 1-5 days <br />at 2O-3OoC, possibly longer depending on <br />specimen volume relative to solution vol- <br />ume and activity or strength of trypsin. <br />Use a volume of trypsin solution at least <br />10 to 40 times the volume of specimens. <br />Completely change trypsin solution every <br />2-3 days. This method is preferred for <br /> <br />21 <br />