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NBT Protocol & Troubleshooting

Product No. 11383213001

Protocol

Possible counterstains in combination with BM Purple (or NBT/BCIP in general):

There are several counterstains possible in combination with BM Purple (or NBT/BCIP in general), including FastGreen FCF and Nuclear Fast Red.

Recommendation for NBT/BCIP or Fast Red preferred substrate for in situ hybridzations:

Fast Red can be recommended for highly expressed genes and when doing double labeling experiments. Information regarding double labeling NBT/BCIP with Fast Red in combination with another fluorescent substrate can be found at: http://www-biology.ucsd.edu/~davek/intro.html

ISH Detection with Fluorescence Microscope Using NBT/BCIP:

Slides stained with NBT/BCIP are usually viewed using a light microscope. Depending on the counterstain, you may also visualize the signals using a microscope equipped with a UV light source. Very faint signals can also be more easily detected using dark field illumination.

Please note that NBT/ BCIP is not a fluorescent precipitate, like the dye Fast Red, which can absorb and emit light at special wavelengths. If you are interested in using a fluorescence signal, combine DIG / AP with the ECF substrate system instead of NBT/BCIP.

Leaving the color reaction overnight:

It is often convenient to leave the color reaction overnight. Such longer times are however strongly dependent on the probe and tissue. First empirically determine if overnight incubation overnight is compatible with your specific probe and tissue. Good post-reaction washing is also important. When performing an overnight color reaction, always keep your coplin jars air tight and in the dark. NBT/BCIP is very sensitive to air; unspecific precipitates will form in the presence of air bubbles. Always check for trapped air bubbles between the slides in your jar and carefully remove them.

Effects when using expired NBT/BCIP for ISH:

When the NBT/BCIP is past its expiration date, it might not be sensitive enough for ISH detection. Please prepare the NBT and the BCIP freshly to achieve highest sensitivity.

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

It is a well known phenomenon that NBT/BCIP is not compatible with classical 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 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.

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 gelatin:

  • 100 ml of 0.2 M phosphate buffer pH 7;
  • Na azide 200 mg;
  • gelatin 15 g, stir until dissolved;
  • glycerol 100 ml.

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

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

Stopping the loss of the strong specific signal when using paraffin–embedded tissue

For best results, perform postfixation of the wholemount embryos after NBT/BCIP detection.

Suggestion: Use 3% paraformaldehyd in PBS buffer supplemented with 1 % glutaraldehyde. This should prevent signal decrease in further steps e.g.,. paraffinization; sectioning etc. Without fixation - the steps where most color signal is washed away are the ethanol wash series and especially the 70% ethanol washing step. The use of xylene should not be the problem. For mounting sections, organic mounting media are recommended, e.g., Merckoglass, Paramount, Euparal.

Troubleshooting

Brown-Purple instead of Blue Signals

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

Weak signals

When the probe is properly evaluated using gel and/or spot assay, it should work well in northern hybridization. For detecting weak signals in ISH, it is important to optimize the quality of the tissue sections, whole mount preparation, as well as 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 beneficial to test a different labeling 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 other tissues may accumulate intracellular lipid droplets. 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 sections using chloroform (10 minutes at RT) prior to the prehybridization procedure.

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

The cause for nonspecific background using NBT/BCIP may be tissue overfixation. 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.

Detection buffer with NBT/BCIP very dark blue with a lot of precipitate

Check the pH of the detection buffer: it must be in the range of pH 9.5, 20 °C. Minimize exposing the detection solution to the air (use air-tight coplin jars). Determine if precipitates are seen in the concentrated NBT/BCIP stock solution. When precipitates occur in the color substrate solution, removed them by warming the solution to +50 °C. If the precipitates do not dissolve, spin the tube prior to use because this may cause background; spinning will not reduce the overall sensitivity.

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 ISH. Please note that slides with NBT/BCIP signals should not be embedded in xylene-based mounting media, because these lead to crystal formation 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 also cause this problem, 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:

  • Assure that the box used for hybridization is well sealed,
  • Perform the hybridization in a humid chamber containing a solution composed of the same buffer and formamide concentration as the hybridization buffer,
  • Cover the sections with coverslips, or more conveniently with parafilm.

Materials

     
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