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75% (or 70%) ethanol solution- In distilled water; if used for specimen preservation (storage). FP, FC Alcian blue stain solution- 20 mg alcian blue per 100 ml of 30% glacial acetic acid in ethanol (solution will keep at room temperature for 3-4 weeks). CS Saturated sodium borate solution- Excess sodium borate powder in dis- tilled water; mix well and allow excess sodium borate to settle; use clear supernatant solution. CS, IC 1 % Potassium hydroxide solution- By weight in distilled water. BL, IC, FC, BS 2% Potassium hydroxide solution- By weight in distilled water; if KOH is used for clearing. IC, FC Bleaching solution- 15% of3 % hydrogen peroxide solution in 1 % KOH solution. BL Trypsin solution- About 0.1-0.2 g (depending on strength or activity level of trypsin) per 100 ml of 30% saturated sodium borate solu- tion in distilled water; mix well but do not allow to froth (Taylor and Van Dyke 1985). Make a fresh solution for each use; it does not keep well. If enzyme clearing used. IC Alizarin red stain solution- Dissolve enough alizarin red powder in 1 % KOH to turn the solution deep pur- ple (about 0.1 g per 100 ml). Or mix about 1 ml of a saturated alizarin red solution per 100 ml of 1 % KOH (satu- rated alizarin red solution is prepared by dissolving excess alizarin red powder in small amount of distilled water, about 1.5-2.0 g per 20 ml). Alizarin red stain solution will keep at least one week. BS 40% glycerin solution- In 1 % KOH (preferred) or distilled water; if glycerin used for storage. FC 70% glycerin solution- In 1 % KOH (preferred) or distilled water; if glycerin used for storage. FC Fixation and preservation 1. Kill and fix specimens in 10% buffered formalin for 24-48 hours. 2. If specimens are to be stored more than a couple days before clearing and staining, preserve them in 3 - 5 % buffered formalin or alcohol (preferably via a graded series of concentrations, e.g., 50% ethanol for 6-24 hours then 75% ethanol). Do not soak in water between fixative and preservative solutions. Cartilage staining procedure 3. Dehydrate formalin-fixed and preserved specimens in 50% ethanol solution for 6-24 hours, then in 100% or absolute ethanol for 12-24 hours. Replace the absolute ethanol and leave at least another 12-24 hours. A more gradual series of alcohol concentra- tions can be used (e.g., 50%, 75%, and 100%), but is usually unnecessary. If speci- mens were preserved in alcohol, skip the 50% ethanol step. For embryos and larvae, dehydration is essential to assure minimal loss of bone while in the acid stain for cartilage. 4. Stain specimens in alcian blue stain solu- tion for 6-24 hours, no longer than neces- sary to adequately stain all cartilage. 5. Rinse specimens in saturated sodium borate solution then soak in fresh saturated sodium borate solution for 6-24 hours to neutralize (change body fluid pH from acid to alkaline) . Bleaching (optional) 6. If specimens are heavily pigmented (such that pigments would obscure desired structures), bleach specimens by placing them in bleaching solution and exposing them to strong light until chromatophore pigment is notably faded, about 20 minutes to a few hours. 24