I have 2 protocols for FISH w/ DNA beacons (approximately 25 nucleotides long) for use on formalin-fixed paraffin embedded tissue (5µm samples) for examination with epifluorescence.

One protocol says to heat the tissue slides to “150 deg C” and then to cool to slides to 90 deg C, layer beacon probe over tissue of interest. Then lock the temperature at the melting temp of the beacon (usually 50-70 deg, but no specific stem melting temps given) and hybridization time 20 min. Bring back to RT. Do 3 consecutive washes in distilled H20. Air dry and examine under Epifluor. 

The other protocol says to layer beacon probe onto tissue area of interest and to cover with hybridization slips and serially heat (no increments given) to 90 deg C for 10 min and then lower temp to 70 deg C for 15 minutes. Bring back to RT. Do 3 consecutive washes in PBS pH 7.0. Air dry and examine under Epifluor.

Questions: The second protocol's denaturation temp looks more reasonable. Would second protocol be better? What would be the increments of serial heating? Can the washes be with distilled H2O instead of PBS?

Thank you!