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Using BM Purple as an alternative AP color reaction substrate
Submit responseIn addition to standard NBT/BCIP, good results were obtained with BM Purple (Roche) under certain circumstances.
Prior to the completion of gastrulation (stage 9), extended NBT/BCIP color reaction often increases background staining in the yolk tissue. For samples that require >3 hours of color reaction, this background staining is a major problem for generating publishable images. In contrast, BM Purple produces almost no background in yolk tissue even after 60 hours of color reaction in room temperature. When BM Purple is used, color reaction could be performed at room temperature or at 37¢XC. It is not recommended to carry out BM Purple color reaction at 4¢XC.
However, BM Purple substrate apparently does not penetrate the tissue very well. It is commendable to run color reaction with NBT/BCIP and BM Purple in parallel for each particular experimental condition in order to determine the best AP substrate for one particular probe in a given developmental stage.
Protocol: 1. Following PBT wash, remove liquid from the tube that contains the specimen. 2. Add an appropriate amount of BM Purple substrate (0.1ml/20 embryos) directly from the bottle to the specimen. 3. Keep the reaction in dark at room temperature or at 37¢XC until the stain is intense enough. As BM Purple reaction progress, cloudy precipitation forms around the specimen and makes it impossible to inspect the specimen. To monitor the color reaction, one can agitate the substrate buffer by gentle pipetting and then remove the substrate buffer after the specimen settle on the bottom. However, since background staining is minimal when BM Purple is used as AP substrate, frequent inspection of specimen during color reaction should be avoided. 4. For many probes, overnight color reaction at room temperature is sufficient. If extended color reaction is needed, replace the substrate every 24 hours. 5. When the desirable stain intensity is reached, stop the color reaction by several washes in PBT. 6. Because the formation of crystal precipitation on the surface of embryos during BM Purple reaction, it is important to wash the specimens with PBT until most of the precipitation has been removed.
