NBT/BCIP Ready-to-Use Tablets Protocol Troubleshooting

BCIP is an alkaline phosphatase (AP) substrate that, after dephosphorylation, is oxidized by NBT to yield a dark-blue indigo precipitating dye. The blue reaction product is insoluble in water. NBT/BCIP is supplied in ready-to-use tablets (Product No. 11697471001).

Troubleshooting the NBT/BCIP Protocol 

Recommended counterstains for non-radioactive in situ hybridization in combination with anti-DIG-AP detection/NBT/BCIP:

NBT/BCIP is not compatible with classicial counterstains. NBT/BCIP signals should not be mounted with xylene-containing mounting media, such as DPX, because these could lead to crystal formations of the color precipitates. Unfortunately, classical counterstains, such as eosin require xylene-containing mounting media.

The following mounting reagents are specifically available for mounting sections with NBT/BCIP signals: Crystalmount from Biomedia or Vectamount or Immunomount from Vector Laboratories. The same companies also offer organic counterstains that 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.

Staining adjacent slides with or without NBT/BCIP detection with a typical counterstain and to mount the slides with the classical xylene-containing mounting medium. This allows the direct comparison of stained tissue with or without signal.

Customer Recommended Protocol* for the Preparation of a mounting medium:

Glycerol gelatine:

• 100 mL of 0.2 M phosphate buffer pH 7;
• Sodium azide 200 mg;
• Gelatine 15 g, stir until dissolved;
• Glycerol 100 mL

Keep mounting medium at 37 °C, add a drop to the slide and coverslip. After hardening of the mounting media the signal lasts for several years without fading of the NBT/BCIP precipitate.

*Note: Customer Recommended Protocols were developed in customer labs and have not been validated by Roche. Roche is not liable for the contents and the successful execution of the protocols. The display of customer protocols is a service of Roche only to facilitate exchange of experience within the research community.

Troubleshooting Weak and Brown Signals during RNA ISH

1. Brown-Purple instead of Blue Signals

For in situ hybridization (ISH), 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.

If a deeper blue or purple signal is desired, try our BM Purple substrate.

2. Weak signals

When the probe is properly tested (gel and/or spot assay were OK), works well in Northern blot, produces weak signals in ISH (provided the protocol is working), it is important to evaluate the quality of the tissue sections, whole mount preparation, or the detection procedure:

• Testing different probe concentrations: Evaluate higher amounts of probe in the hybridization mix: e.g. 1, 2, 4, 8 μL of the 50 μL diluted standard reaction per 50 μL hybridization solution.
• Include a strong positive control
• Test an alternative protocol using chloroform instead of Proteinase K, glycine.

It may also be worthwhile to test a completely different protocol.

Note: For DIG in situ hybridization, it is important that there is no drying out of the slides after the pretreatment steps and prior to the hybridization step. Drying of the slides can causes nonspecific background.

Nonspecific “vesicular” blue background after performing the alkaline phosphate (NBT/BCIP) detection procedure with heart samples for ISH:

Heart, and also some other tissues, may have accumulated lipid droplets intracellularly. When using the alkaline phosphate (NBT/BCIP) detection procedure on cryosections some of the color precipitate is trapped in these lipid droplets. This problem is solved by delipidizing the sections in chloroform (10 minutes at RT) before the prehybridization procedure.

High generalized blue background after using the normal alkaline phosphatase (NBT/BCIP) detection system:

The cause for high generalized nonspecific background using NBT/BCIP may be overfixation of the tissue. This usually results in a generalized blue staining of the entire tissue. It may not be possible to avoid this–because the tissue you are using is from a source you cannot change–this background should however not interfere with producing the specific signal.

Recommendations to prevent fading of DIG NBT/BCIP signals for in situ hybridization experiments:

It is unusual for fading of NBT/BCIP to be observed after in situ hybridization. Usually fading is an issue for fluorescence in situ hybridization.

Slides with NBT/BCIP signals should never be embedded in xylene-based mounting media because these could lead to crystal formations of the color precipitates.

Nonspecific Background at Section borders:

• Make sure that the sections do not dry out during the detection procedure. In addition to the detection procedure, which may cause this type of background, other steps may account for the problem described, such as the hybridization procedure.
• Make sure that the section is fully covered with prehybridization/hybridization buffer during hybridization. A reduction of the volume and drying out of the sections during these steps may be the underlying reason. For best results:
  - assuring that the box used for hybridization is well sealed,
  - performing the hybridization in a humid chamber containing a solution composed of the same buffer and formamide concentration as the hybridization buffer,
  - covering the sections with coverslips, or more conveniently with parafilm.