1. 20X SSC stock buffer: 3 M NaCl, 0.3 M Na3citrate, pH 7.0.

2. Denaturing solution: 70% formamide (FA; Invitrogen), 2X standard saline citrate (SSC), pH 7.0. Store at 4°C.

3. Hybridization master mix (MM2.1): 14.3% (w/v) dextran sulfate, 78.6% FA, 2.9X SSC, pH 7.0. For 10 mL MM2.1, mix 1.45 mL of 20X SSC with 0.7 mL water, dissolve 1.43 g dextran sulfate (Calbiochem, San Diego, CA), incubate overnight, and then add 7.86 mL FA (Invitrogen). Aliquot in 1.5-mL microcentrifuge tubes and store at -20°C.

4. Maleic acid buffer: 100 mM maleic acid, 150 mM NaCl; adjust to pH 7.5 with concentrated NaOH.

5. Blocking solutions.

a. Blocking stock solution: dissolve blocking reagent (Roche Molecular Biochem-icals) in 10% (w/v) maleic acid buffer with shaking and heating. Autoclave stock solution and store in aliquots at 4°C.

b. Slide blocking solution: 5X SSC containing 2% blocking reagent and 0.1% N-lauroyl sarcosine. Combine 0.05 g N-lauroyl sarcosine (Na salt) and 1 g blocking reagent with 12.5 mL of 20X SSC, pH 7.0, and add 30 mL water. Heat to 60°C while stirring and bring the final volume to 50 mL with water, when the blocking reagent is dissolved. Aliquot into 1.5-mL tubes, spin at 300g for 10 min, and store at 4°C.

6. PN buffer: 0.1 M sodium phosphate, pH 8.0, 0.1% Nonidet-P40 in water. This buffer is prepared by dissolving 26.8 g of Na2HPO4 (dibasic) in 1 L water and 8.2794 g of Na2HPO4 (monobasic) in 600 mL water. Note the amount of monobasic solution added. Tirate the dibasic solution (pH > 9) by adding small volumes of the monobasic solution (pH ~4.5) until a pH of 8.0 is reached. Add Nonidet-P40 to 0.05% (v/v).

7. PNM buffer: dissolve 5 g of nonfat dry milk (Carnation, Wilkes-Barre, PA) in 100 mL of PN buffer, incubate at 50°C overnight, add 1/50 vol sodium azide, spin at 1000g for 30 min, aliquot clear supernatant into 1.5-mL tubes, and store at 4°C. Spin at 2000g for 30 s prior to use of the clear supernatant.

8. Rhodamine-conjugated antibodies against digoxigenin, made in sheep (Roche Molecular Biochemicals), mouse-derived antibodies against FITC (DAKO, Carpintera, CA), FITC-conjugated antimouse antibodies made in horse (Vector, Burlingame, CA), and biotinylated anti-avidin antibodies made in goat (Vector) are prepared as stock solutions (1 mg/mL) in PNM, stored at 4°C, and diluted 1:50 with PNM just prior to use. Store at 4°C. Avidin conjugated to AMCA or FITC (Vector): stock solution is 2 mg/mL in PNM, diluted 1:500 prior to use. Store at 4°C.

9. Antifade solution: 1% p-phenylenediamine, 15 mM NaCl, 1 mM H2PO4, pH 8.0, 90% glycerol. Store in 1-mL aliquots at -80°C, and keep one aliquot at -20°C for everyday use (see Note 4).

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