1. This protocol works well on intact embryos up to at least mouse embryonic d 12, chick Hamburger and Hamilton (3) stage 24, and zebrafish to 72 h. For older embryos, it is likely that some block dissection will be required prior to fixation to keep background levels low. Alternatively, some tissues, such as the neural tube, can be dissected after performing the procedure on intact older embryos.

2. It is worth trying a range of fixatives before ruling out the suitability of an antibody or antiserum for this procedure. In addition to standard paraformaldehyde fixation (Subheading 3., step 1), it is worth trying 0.5% (w/v) paraformaldehyde, 0.5% (v/v) glutaraldehyde; 4% (w/v) paraformaldehyde, 0.1% (v/v) glut-araldehyde; 5% (v/v) acetone, 95% (v/v) ethanol; perfix (Fisher Scientific, Loughborough, UK); dry AnalaR acetone. The latter three fixatives are precipitating (rather than crosslinking) fixatives and, thus, are probably not worth trying if the antigen is a small soluble protein (e.g., Mr < 20,000). For a detailed consideration of antibody-antigen interactions including fixation, see ref. 4.

3. Some antibodies may bind more strongly at 37°C, and for these the use of a combined rotating wheel and incubator, such as a "Spin 'n' Stack" (Hybaid, Teddington, UK), is recommended.

4. Required dilution must be determined empirically and will depend on titer and affinity. We have used dilutions varying between 1:2 and 1:500 for MAbs raised against different antigens.

5. The diluted antibody can be frequently reused one or more times. It should be retrieved and stored at 4°C between uses.

6. DAB is carcinogenic. All materials in contact with it and all waste solutions should be treated overnight with excess bleach to break it down.

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