1. For larger embryos, some procedures, particularly washes after hybridization and after incubation with antibodies, are probably more efficiently performed in an incubator containing a slowly rotating wheel, e.g., "Spin 'n' Stack" (Hybaid).

2. If possible, it is worth opening the rostral neural tubes (e.g., the midbrain vesicle or the forebrain) of mouse and chick embryos older than E9.5 (mouse) and HH stage 14 chick to help reduce probe or antibody entrapment; any structure with a large lumen can pose this problem (e.g., also heart and gut) and may be worth dissecting for separate hybridization. As embryos get larger, the problems of high background and poor penetration increase, and it is advisable to dissect further embryos larger than E5.5 (chicks) and E11.5 (mice) into smaller pieces. If a particular tissue is of interest, results are considerably enhanced if it can be exposed or dissected free of surrounding tissue prior to fixation and subsequent processing. Such approaches have allowed the technique to be extended to late stages of development (see, e.g., ref. 5).

3. Fixation times will vary with the size of the embryo from a few hours (mice up to E10.5, chicks to HH stage 20) to 12 h or longer. In our experience, fixation for 23 d does not adversely affect results, but thereafter there is increasing reduction in sensitivity.

4. Some protocols suggest that embryos can be stored for a few months in methanol at this stage. However, it is our experience that this results in considerable loss of sensitivity, and we advocate long-term storage (>1 wk) at -20°C in prehybridization solution.

5. The optimum time for proteinase K treatment ideally should be determined for each new batch of protease. However, treatment for 7 min is a good starting point. For larger embryos, the position of the tissue of interest should be taken into consideration: peripheral tissues (e.g., epidermis, dermis) and small appendages (e.g., developing limbs) require shorter incubation times than deeper body organs. For very young zebrafish embryos (<10 somites), protein-ase K treatment may not be required at all.

6. Embryos become very fragile after this step until after they are postfixed and must be treated very gently.

7. Embryos can be stored for months at -20°C in prehybridization solution before or after incubating at 70°C; precipitation of SDS from the buffer has no effect on the preservation of embryos or RNA under these conditions.

8. Restriction enzyme sites should be selected to produce a transcript of 300-1000 bp. At any larger size, nonspecific background signal occurs in embryos probably owing to trapping of probe. Also, if possible, select enzymes that produce a blunt end or a 5'- overhang, because 3'- overhanging ends facilitate "wraparound" transcripts, resulting from the polymerase transcribing back along the complementary DNA strand. If there is no alternative to using an enzyme producing a 3'-overhanging end, the following procedure can be used after digestion to "end polish," generating a blunt end. After digestion, add Klenow polymerase to a final concentration of 5 U/pg DNA template, and incubate at 22°C for 15 min prior to proceeding with the rest of the transcription protocol.

9. Either DIG- or FITC-labeled probes are suitable, and if expression of two genes is to be analyzed simultaneously, one probe is made with each. However, the signal produced from an FITC-labeled probe is weaker than that from the same probe labeled with DIG. Thus, when performing hybridizations with two probes, the probe for the most abundant of the mRNAs of interest generally should be FITC-labeled. In our experience, FITC-labeled probes should be coupled with the fast red substrate system (Subheading 3.6.) and DIG-labeled probes with the NBT/BCIP system to produce the most sensitive results. However, the red product of the fast red system cannot be visualized over the dark blue product of the NBT/BCIP system, and this must be taken into account when deciding which to label with DIG or FITC if the two genes being studied are expressed in the same tissue or organ.

10. The second precipitation can frequently be omitted but it helps to ensure that unincorporated nucleotides are efficiently removed, since their presence increases nonspecific "background" signal. This is especially true of FITC-UTP-labeled probes which sometimes benefit from a third precipitation.

11. The RNase treatment (Subheading 3.5., steps 2-5) should be omitted if probes are derived from DNA sequences of a different species. Frequently, it can be omitted anyway, but it should be borne in mind that its inclusion probably reduces overall background levels and the possibility of crosshybridization to transcripts derived from closely related members of a multigene family. For example, expression of transcripts of the vertebrate engrailed genes, En-1 and En-2, can only be distinguished if RNase treatment is included (C. Logan, personal communication).

12. In our experience, embryo powder prepared from a single vertebrate species can be used to prevent nonspecific binding of antibody to all vertebrate classes.

13. Blocking reagent (Boehringer Mannheim; BM 1096 176) can be used instead of embryo powder. Blocking reagent is made up as a 10% (w/v) stock in 1X MABT by heating to dissolve it, and then autoclaving, aliquoting, and freezing. 10X MABT is 500 mM maleic acid, 750 mM NaCl pH 7.5, and 0.5% (v/v) Tween-20, and 1 L is prepared by dissolving 58 g maleic acid, 44 g NaCl, and 35 g NaOH (the NaOH is required to dissolve the maleic acid). The pH is adjusted to 7.5 with 10 N NaOH, and 5 mL Tween -20 is added. The dissolved blocking reagent is stored in aliquots at -20°C and used at 2% final concentration instead of embryo powder.

14. If embryos are to be sectioned, we have found that it is best to "overdevelop" the apparent level of signal on whole embryos in order to obtain sufficient intensity on sections.

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