Materials 21 Chemicals

1. Heptane-« (Sigma-Aldrich). Wear gloves and handle with care; heptane is both an irritant and highly flammable!

Methanol (Sigma-Aldrich, protein sequencing grade). Wear gloves and handle with care. Methanol is toxic and highly flammable! Keep aliquots at room temperature and at -20°C.

Acetone (Sigma-Aldrich, 100% solution, ACS reagent). Wear gloves and handle with care. Acetone is an irritant and highly flammable! Keep aliquots at -20°C. Dimethyl sulfoxide (DMSO; Sigma-Aldrich, ACS reagent). Make aliquots of 500 |L and store them at -20°C.

H2O2 (Hydrogen peroxide, Sigma-Aldrich, 30% solution). Wear gloves and handle with care! Keep cold and use fresh.

Fetal calf serum (FCS) (Invitrogen). Make aliquots of 500 |L and store them at -20°C. TOTO-3 (Molecular Probes, Eugene, OR). Make aliquots of 10 |L and store them at -20°C.

Propidium iodide (Fluka). Make a 1-mg/mL stock solution in water. Wear gloves and handle in a chemical fume hood. Store in a cool dry place and keep tightly closed.

Vectashield mounting medium (Vector Laboratories). Keep refrigerated in the dark. Drosophila Schneider's medium (Invitrogen).

Solutions

EBR stock solution: 130 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 10 mM HEPES, pH 6.9. Store at room temperature for up to 2-3 mo.

Buffer B stock solution: 100 mM KH2PO4/K2HPO4 pH 6.8, 150 mM NaCl, 450 mM KCl, 20 mM MgCl26H2O.

Buffer B fixative solution: 1 vol of 37% formaldehyde (Sigma-Aldrich, molecular-biology grade containing 10-15% methanol), 1 vol of buffer B and 4 vol of distilled H2O. Store the 37% formaldehyde stock solution at room temperature. It is unstable, tending to polymerize in contact with air. Keep the cap of the bottle tight. If the solution is clear and no precipitate formed, it can be used for up to 6 mo. Wear gloves and work in a fume hood, as the formaldehyde vapor is toxic! 10% Paraformaldehyde (PP) (Polysciences, EM grade) in phosphate-buffered saline (PBS): To prepare 50 mL, weight out 5 g of PP into 40 mL PBS. Dissolve the PP by warming the solution to 60°C. Do not overheat! If the solution turns brownish, discard it and start again! If the PP does not dissolve relatively easily to give a clear solution, then add 1 mL of 1 N NaOH, and this should help to get the PP dissolved. Adjust to pH 7 with 1 N HCl. Bring the final volume of the solution to 50 mL with PBS and filter-sterilize half of the solution using 0.2-|im filter units. Store the filtered solution in a tightly capped bottle at room temperature and use within 1-2 d, or make frozen aliquots of 500 |L, which are fairly stable for 6 mo. Wear gloves and work in a fume hood, as the PP powder and vapor are toxic!

Phosphate-buffered saline (PBS) solution: Dissolve 1 PBS tablet (Sigma) in 200 mL of distilled H2O.

PBT solution: PBS + 0.1% Triton X-100 (Sigma, molecular-biology grade).

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