Staining protocols for both lacZ and AP reporter gene activity are presented below. For embryonic stages up to 13.5 days postcoitum (dpc), embryos may be stained intact. After this age, problems arise due to the developing skin, which acts as a barrier to fixation and staining. Older embryos may be partially dissected or processed as frozen sections to overcome this (7).
1. Dissect embryos out of uteri and decidua and free of extraembryonic membranes in PBS. Retain the placentas for transgenic identification if necessary (see Subheading 3.5.). Rinse the embryos with PBS to remove serum (see Notes 21 and 22).
2. Transfer the embryos into fixative (see Notes 23 and 24). Fixation time depends on the size of the embryo. Ten to 15 min are sufficient for embryos up to 8.5 dpc, 15-30 min for 8.5-11.5 dpc and up to 90 min for 14.5 dpc embryos.
3. Wash the fixed embryos twice in a large volume of embryo wash for 20 min each at room temperature. Proceed with the appropriate staining method as described in Subheadings 3.4.1. and 3.4.2.
Two possible substrates are available for fi-galactosidase staining (see Table 2). 5-bromo-4-chloro-3-indolyl-fi-D-galactopyranoside (X-gal) was the first chromogenic substrate available for the detection of B-galactosidase activity and is still the most widely used. 5-bromoindolyl-fi-O-galacto-pyranoside (Bluo-gal) is a more recent development and has some merits over X-gal for certain applications. The advantages of X-gal are that it gives a light-blue reaction product that is more photogenic than the dark-blue of Bluo-gal in whole-mount embryos, and it is also cheaper. It does have some drawbacks, however, particularly in the prolonged staining of embryos older than 11.5 dpc, where levels of endogenous fi-galactosidase activity increase and may lead to a greenish-blue background staining (see Note 25). This problem does not seem to arise when staining embryos with Bluo-gal, and it is particularly useful when dealing with weak expression at these stages. The other advantages of Bluo-gal over X-gal are in the histological examination of stained tissue sections where greater sensitivity and morphological resolution can be obtained with the use of hematoxylin and eosin (H&E) counter-staining methods (49).
1. X-gal staining: Incubate the fixed and washed embryos in X-gal stain solution in the dark. Staining conditions depend on the levels of P-galactosidase activity and on the size of the samples (see Note 26). Maximal staining is usually seen after 16-24 h. Embryos of ages up to 10.5 dpc can be stained at 37°C, whereas older embryos should be stained at lower temperatures (30°C or room temperature).
2. Bluo-gal staining: Incubate in Bluo-gal stain solution. The conditions are as for X-gal staining. However, higher temperatures can be used with older embryos owing to the reduced problem of background staining.
Several possible chromogenic substrates producing a range of colors are available for AP detection (Table 2). BCIP/NBT (e.g., Boehringer, Sigma) is the most commonly used substrate, producing a blue-purple color, and is suitable for most applications. BM Purple (Boehringer), which gives a deep purple color, is more expensive than BCIP/NBT but has a greater sensitivity and produces less background staining. The purple colors of these substrates may not contrast well with blue when also staining for P-galactosidase activity. In this case, red (Napthol-AS-TR-phosphate (NATP)/Fast Red RC) or green (Napthol-AS-GR-phosphate/Fast Blue BN) color producing substrates may prove useful (Boehringer, Sigma). For double staining proceedures the P-galactosidase step should be performed first before proceeding with the alkaline phosphatase protocol, as heat treatment will inactivate the P-galac-tosidase enzyme (20).
1. Incubate the fixed, washed embryos at 65°C in PBS for 10-45 min to preferentially reduce endogenous AP activity (see Note 27).
2. Stain the embryos in AP stain solution at room temperature in the dark.
3. Terminate the staining reaction by immersing the embryos in PBS containing 20 mM EDTA.
3.4.3. Poststaining Treatment of Samples
1. Wash the embryos for 10 min in two changes of PBS to remove any stain solution.
2. If samples are to be sectioned, postfix in Mirsky's or 4% (w/v) paraformaldehyde prior to embedding.
3. Store at 4°C in 70% (v/v) ethanol after dehydration through a series of 30% (v/v) and 50% (v/v) ethanol (see Note 28).
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