Chwen-Huey Wu's method.  The chromosomes are incubated in TBST (150 mM NaCl, 10 mM Tris-Cl pH 7.5, 0.03% Triton) plus 5% powdered milk.

Chromosome are squashed according to our usual protocol but the finished slide is immersed in overnight at 4^oC in TBST + 5% powdered milk.

The next day, the slides are rinsed briefly in TBST.

Primary antibody is diluted in TBST + 10% Fetal Bovine Serum and incubated on the slide at 4^oC for 2 hours.

Wash slides 2 times for 5 to 10 minutes each with TBST.

Secondary antibody is diluted in TBST + 10% Fetal Bovine Serum and incubated on the slide at 4^oC for 2 hours.

Wash slide with TBST containing 10 ng/ml Hoechst for 10 to 20 minutes
Wash slide with TBST lacking Hoechst for 10 to 20 minutes.

Mount the slides with 100 mM Tris-Cl pH 8.5, 80% glycerol, 2% n-propylgallate or with Vectashield H-1000 mounting medium from Vector.