A Copper Sulfate-Silver Nitrate Method for Nerve Fibers of Planarians

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Abstract
For staining in toto, planarians are fixed in a mixture of 10 ml of commercial formalin, 45 ml of 95% ethanol and 2 ml of glacial acetic acid. After treatment with 70% ethanol 3-10 days, they are washed in distilled water and immersed in 10% CuSO4. 5H2O for 3 hr at 50° C, transferred without washing to 1% AgNO3 for 1.0-1.5 hr at 50° C; and then developed in: 10 ml of 1% pyrogallol, 100 ml of 56% ethanol and 1 ml of 0.2% nitric acid. Gold toning, 5% Na2S2O3 and dehydration follow as usual. For staining sections, material is fixed in the same fixative, embedded in paraffin and sectioned at 10 μ. After bringing sections to water, they are immersed in 20% CuSO4. 5H2O for 48 hr at 37° C; then rinsed briefly in distilled water and placed in 7% AgNO3 for 24 hr at 37° C. They are washed briefly in distilled water and reduced in: hydroquincne, 1 gm; Na2SO3, 5 gm and distilled water 100 ml. Gold toning, followed by 5% Na2S2O3 and dehydration completes the process. Any counterstaining may follow.