An alternative to the processing steps listed in Subheading 3.4. can be used. For most markers that are routinely assayed, this shortened procedure works well and requires much less effort. After pretreatment and hybridization as described above, process the samples as follows:
1. Replace hybridization solution with 50% formamide/5X SSC for 10 min at 60°C.
2. Wash once in 25% formamide/2X SSC for 10 min at 60°C.
3. Wash twice for 30 min with 2X SSC/0.1% CHAPS at 60°C.
4. Wash twice for 30 min with 0.2X SSC/0.1% CHAPS at 60°C.
5. After the final wash at 60°C, wash twice at room temperature with PBT or MAb, and then proceed to the blocking step and antibody incubations as described above (Subheading 3.5.).
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