Q: Why not stop the Proteinase K reaction with glycine?
A: Glycine inhibits the enzyme and stops the digestion immediately. Repeated washing in PBT accomplishes the same thing more gradually. In different protocols, greatly varying final ProtK concentrations (4-50 µg/ml) and incubation times (1-10 minutes) are used. In general, higher concentrations require shorter incubation times and the use of a glycine buffer to stop the reaction. If the reaction time is short, the margin of error is smaller and one will have more control over stopping the digestion at the precise time by using glycine. With lower ProtK concentrations and longer incubation times, the timing of stopping the digestion is not so critical and the gradual removal of the enzyme with repeated washes is sufficient.
Q: Different manufacturers produce ProtK that is measured either in units of activity/ml or mg/ml. Does this matter?
A: It is not important whether ProtK is provided in units/ml or mg/ml, as long as the activity is known for this application. The stated 'units' do not measure digestion activity on fixed Drosophila embryos and the optimum performance conditions for any stock of ProtK must be determined by testing. On the other hand, the indicated units provide a good estimate on where to start in the titration of a stock, particularly if one is switching brands. One last comment about using ProtK: it definitely increases the absolute signal level of these FISH embryos, as well as reduces the background. It is worth the extra trouble.
Q: Can PCR be used to generate probe templates? This eliminates the need for growing large preps of plasmid DNA.
A: Roche offers a PCR protocol for making labeled probes, and the BDGP gene expression project uses a PCR protocol for its high-throughput embryo staining. We have used probes synthesized from PCR-generated templates successfully in this protocol.
Q: Why not use DEPC-treated ddH2O?
A: We have not experienced problems in obtaining strong signals and therefore have left out this precaution. However, RNase contamination can severely reduce the quality of ISH stains and DEPC treatment of solutions is a good countermeasure. We recommend the commonly available non-DEPC treated, filtered, certified nuclease-free ddH2O in the probe synthesis reactions.
Q: Why are the probes fragmented? Some protocols claim that this is unnecessary and actually makes probes worse in some cases.
A: We have not yet addressed this question systematically, but have observed that some full-length, non-hydrolyzed probes produce very high background, while the same probes hydrolyzed give no background. We estimate that between 0.5-1 kb probe size, fragmentation becomes necessary.
Q: Sometimes after the hybridization steps clumps of embryos form that never break up. Why?
A: This clumping is most likely due to excessive Proteinase K treatment.
Q: This protocol includes a very long hybridization step. Other protocols have only 12-18 hour hybridizations. Does the quality of the in situ increase appreciably with those extra hours?
A: In 50% formamide, probes come to equilibrium very slowly with their proper targets, while being prevented from forming stable hybrids with their mismatch targets. Others have confirmed that longer hybridization times can dramatically improve the performance of probes that before gave weak stains. In these cases, the hybridization time was extended from one overnight (12-16 hours) to two (36-40 hours).
Q: Why is 55° C. used for the hybridization temperature? Other protocols hybridize at 65° C.
A: The original whole mount non-radioactive in situ hybridization technique, described in 1989 by Tautz and Pfeifle, employed labeled DNA probes and hybridization conditions that very closely resemble those used for DNA:DNA filter hybridizations: 50% formamide and 45° C. In the embryo, DNA:RNA hybrids were formed and the same hybridization conditions were used successfully. Shortly thereafter, RNA probes came into use, and the rule of thumb, that for a given sequence, the melting temperature of the RNA:RNA hybrid was 10° C. higher than the corresponding DNA:DNA hybrid, is behind the increased 55° C. temperature for RNA probes. It is not clear whether using yet higher temperatures (60-70° C.) would improve signals, but it might help in some cases of unacceptably high background staining caused by non-specific binding of probes to the embryos.