1. Limited alkaline hydrolysis of the probe to reduce its overall length to 200-300 bp is a consideration. Most probes work well when used as full length (2-3 kb probes have worked well), but others may function better if their length is reduced. To hydrolyze a probe, resuspend the precipitated probe in 50 pL of a solution containing 40 mM sodium bicarbonate, and 60 mM sodium carbonate. Heat at 60°C for 30 min to 1 h. After hydrolysis, increase the volume to 200 pL with water and precipitate. Redissolve the pellet in water, and use at a final concentration of 1 pg/mL for hybridization.

2. If persistent background is a problem, consider preincubating the anti-digoxygenin antibody with embryonic acetone powder prior to use. Acetone powder is easy to make and the protocol can be found in Antibodies: A Laboratory Manual (7). Additionally, background is usually reduced when BM purple AP substrate is used for the color development instead of NBT/BCIP in APB.

3. Whole-mount specimens can be processed for histological sectioning, which allows more detailed analysis. If sectioning is desired, it is a good idea to develop the color as intensely as possible (overstain), monitoring to ensure that background does not become a problem. It is essential to postfix the tissue to stabilize the stain before processing for histology. To prepare the tissues for sectioning, dehydrate through a series of methanol (30, 50, 70, 90, and 100%), and clear with xylene or histoclear. Embed tissues in wax, and section at 10-20 pm. Thicker sections provide more intense signal. Tissues can be counterstained with eosin, which provides a nice contrast to the purple color reaction product.

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