NBT Crystals Protocol Troubleshooting

Nitro blue tetrazolium (NBT) chloride crystals (Product No. 11585029001) combined with 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) is ideal for the sensitive detection of alkaline phosphatase (AP) in blotting protocols, including Southern blot, Northern blot, Western blot, colony and plaque lifts, in situ hybridization, and for applications in immunohistochemistry and immunocytochemistry. NBT/BCIP may be used with both nitrocellulose and nylon membranes.

Protocol Troubleshooting

Counterstains

• There are several counterstains possible in combination with BM Purple (or NBT/BCIP in general), for instance, FastGreen FCF or Nuclear Fast Red. For details, see the protocol described in the Roche DIG Application Manual for Nonradioactive in situ Hybridization.

Mounting Media

• NBT/BCIP signals should not be mounted with xylene containing mounting media (e.g., DPX), because these could lead to crystal formations of the color precipitates. Classical counterstains, such as eosin, however, require xylene containing mounting media.
• The following mounting reagents are specifically on the market for mounting sections with NBT/BCIP signals: Crystalmount from Biomedia or Vectamount or Immunomount from Vector Laboratories. The same companies also offer organic counterstains which are compatible with these mounting media (e.g., Vector Methyl Green, Vector Nuclear Fast Red).
• The results of combining a particular counterstain with any of the mounting media primarily depends on the type of tissue used for NBT/BCIP color detection.
• Stain adjacent slides with or without NBT/BCIP detection with a typical counterstain and mount the slides with the classical xylene containing mounting medium: This allows the direct comparison of stained tissue with or without signal.
• Some tissues, e.g., heart sections, may accumulate lipid droplets intracellularly. When using the alkaline phosphate (NBT/BCIP) detection procedure on cryosections, some of the color precipitate can be trapped in these lipid droplets. This problem is solved by delipidizing these sections in chloroform (10 minutes at RT) prior to the prehybridization procedure.

Precipitates

• Precipitates in the stock solution can be dissolved by warming and gentle shaking. Before pipetting, spin down and remove from the top layer.
• pH of the detection buffer must be in the range of pH 9.5, (at 20 °C) and exposition of the detection solution to the air must be minimized (air-tight coplin jars).

High General Blue Background

• High general non-specific background using NBT/BCIP may be due to overfixation of the tissue. This usually results in a general blue staining of the whole tissue. This background does not interfere with the specific signal.

Brown-Purple Instead of Blue Signals

• With in situ hybridization the color of NBT/BCIP precipitate can vary from blue to brown or purple. The final color depends primarily on the abundance of target mRNA in the tissue, but is also influenced by probe length and labeling intensity. The pH of the detection solution (reaction buffer for alkaline phosphatase) can also play a role and should be carefully adjusted to pH 9.5.
• In general, the more abundant the target RNA, the stronger the corresponding signal, resulting in a deeper blue color precipitate.
• A deep blue or purple signal is seen with BM Purple substrate.
• Drying of slides from the edges may cause unspecific background.