[Przejdź do zawartości](https://www.sigmaaldrich.com#main-content) [](https://www.sigmaaldrich.com/PL/pl) Produkty Koszyk0 PLPL Produkty ProduktyZastosowaniaUsługiZasobyWsparcie [Zaloguj się / Zarejestruj się](https://www.sigmaaldrich.com/oidc-sign-in) [Wyszukiwanie zamówienia](https://www.sigmaaldrich.com/PL/pl/order-lookup) [Szybkie zamówienie](https://www.sigmaaldrich.com/PL/pl/quick-order) Koszyk0 [Home](https://www.sigmaaldrich.com/PL/en)[Nucleic Acid Labeling & Detection](https://www.sigmaaldrich.com/PL/en/applications/genomics/nucleic-acid-labeling-and-detection)NBT Protocol & Troubleshooting # NBT Protocol & Troubleshooting Product No. [11383213001](https://www.sigmaaldrich.com/PL/en/product/roche/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](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., M-Glas®, 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 Przepraszamy, wystąpił nieoczekiwany błąd Response not successful: Received status code 500 __Related Articles__ - [DIG Easy Hyb™ Granules Protocol & Troubleshooting](https://www.sigmaaldrich.com/PL/en/technical-documents/protocol/genomics/nucleic-acid-labeling-and-detection/dig-easy-hyb-granules) - [DIG Northern Starter Kit Protocol & Troubleshooting](https://www.sigmaaldrich.com/PL/en/technical-documents/protocol/genomics/nucleic-acid-labeling-and-detection/dig-northern-starter-kit) - [DIG RNA Labeling Kit (SP6/T7) Protocol & Troubleshooting](https://www.sigmaaldrich.com/PL/en/technical-documents/protocol/genomics/nucleic-acid-labeling-and-detection/dig-rna-labeling-kit) - [DIG RNA Labeling Mix Protocol Troubleshooting](https://www.sigmaaldrich.com/PL/en/technical-documents/protocol/genomics/nucleic-acid-labeling-and-detection/dig-rna-labeling-mix) - [NBT/BCIP Ready-to-Use Tablets Protocol Troubleshooting](https://www.sigmaaldrich.com/PL/en/technical-documents/protocol/genomics/nucleic-acid-labeling-and-detection/nbt-bcip-ready-to-use-tablets) - [NBT/BCIP Stock Solution Protocol & Troubleshooting](https://www.sigmaaldrich.com/PL/en/technical-documents/protocol/genomics/nucleic-acid-labeling-and-detection/nbt-bcip-stock-solution) - [NBT Crystals Protocol Troubleshooting](https://www.sigmaaldrich.com/PL/en/technical-documents/protocol/genomics/nucleic-acid-labeling-and-detection/nbt-crystals) - [Oligonucleotide Melting Temperature](https://www.sigmaaldrich.com/PL/en/technical-documents/protocol/genomics/pcr/oligos-melting-temp) __Related Product Categories__ - [Roche Life Science Products](https://www.sigmaaldrich.com/PL/en/products/molecular-biology-and-functional-genomics/roche-life-science-products) - [Allergenic Fragrance Testing – Certified Materials](https://www.sigmaaldrich.com/PL/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/allergenic-fragrance-testing) - [Analysis of Alkylphenols Using New Internal Standards](https://www.sigmaaldrich.com/PL/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/analysis-of-alkylphenols) - [FAME Standard for Optimizing GC System Performance](https://www.sigmaaldrich.com/PL/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/fame-standard-for) - [Fast and Reliable Environmental Analysis of Aldehyde and Ketone Air Pollutants](https://www.sigmaaldrich.com/PL/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/fast-and-reliable) - [Hydrocarbon Oil Index in Samples](https://www.sigmaaldrich.com/PL/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/hydrocarbon-oil-index) - [Neonicotinoids](https://www.sigmaaldrich.com/PL/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/neonicotinoids-bee-pesticides) - [New GC-MS Method for Mycotoxin Analysis](https://www.sigmaaldrich.com/PL/en/technical-documents/protocol/analytical-chemistry/calibration-qualification-and-validation/new-gc-ms-method-for-mycotoxin-analysis) Góra __Zaloguj się, aby kontynuować__ Zaloguj się lub utwórz konto, aby kontynuować. Zaloguj się__Nie masz konta użytkownika?__Zarejestruj Wsparcie[Obsługa klienta](https://www.sigmaaldrich.com/PL/pl/support/customer-support)[Kontakt](https://www.sigmaaldrich.com/PL/pl/collections/offices)[FAQ](https://maestro.my.site.com/knowledgeportal/s/)[Karty charakterystyki (SDS)](https://www.sigmaaldrich.com/PL/pl/documents-search?tab=sds)[Certyfikaty (COA/COO)](https://www.sigmaaldrich.com/PL/pl/documents-search?tab=coa)[Jakość i przepisy](https://www.sigmaaldrich.com/PL/pl/life-science/quality-and-regulatory-management)[Kalkulatory i aplikacje](https://www.sigmaaldrich.com/PL/pl/support/calculators-and-apps)[Webinaria](https://www.sigmaaldrich.com/PL/pl/collections/webinars) Zamówienia[Wyszukiwanie zamówień](https://www.sigmaaldrich.com/PL/pl/order-lookup)[Szybkie zamówienie](https://www.sigmaaldrich.com/PL/pl/quick-order)[Produkty niestandardowe](https://www.sigmaaldrich.com/PL/pl/services/custom-products)[Rozwiązania eCommerce](https://www.sigmaaldrich.com/PL/pl/life-science/ecommerce/ecommerce-solutions) Firma[O nas](https://www.sigmaaldrich.com/PL/pl/life-science/about-us)[Odpowiedzialność](https://www.sigmaaldrich.com/PL/pl/life-science/ssbi)[Wydarzenia](https://www.sigmaaldrich.com/PL/pl/collections/events)[Komunikaty prasowe](https://www.sigmaaldrich.com/PL/pl/collections/press)[Programy](https://www.sigmaaldrich.com/PL/pl/life-science/partnership-programs)[Kariera](https://careers.merckgroup.com/global/en)[Biura](https://www.sigmaaldrich.com/PL/pl/collections/offices) Media społecznościowe [](https://www.linkedin.com/company/merck-life-science) [](https://x.com/Merck_lifesci) [](https://www.facebook.com/Merck.lifescience) [](https://www.instagram.com/mercklifescience/) [](https://www.youtube.com/@MerckLifeScience/featured) Merck __Badania. Rozwój. Produkcja.__ Jesteśmy wiodącym dostawcą dla globalnej branży Life Science, oferującym rozwiązania i usługi w zakresie badań, rozwoju i produkcji biotechnologicznej oraz rozwoju i produkcji leków farmaceutycznych. [](https://www.sigmaaldrich.com/PL/pl) [Rozwiązania Sigma-Aldrich®](https://www.sigmaaldrich.com/PL/pl/life-science/sigma-aldrich)[Rozwiązania BioReliance®](https://www.sigmaaldrich.com/PL/pl/life-science/bioreliance)[Roztwory Millipore®](https://www.sigmaaldrich.com/PL/pl/life-science/millipore)[Rozwiązania SAFC®](https://www.sigmaaldrich.com/PL/pl/life-science/safc)[Rozwiązania Milli-Q®](https://www.sigmaaldrich.com/PL/pl/life-science/milliq)[Rozwiązania Supelco®](https://www.sigmaaldrich.com/PL/pl/life-science/supelco) © 2026 Merck KGaA, Darmstadt, Niemcy i/lub jej podmioty stowarzyszone. Wszelkie prawa zastrzeżone, w tym eksploracja tekstu i danych na potrzeby szkolenia w zakresie sztucznej inteligencji i podobnych technologii. Powielanie jakichkolwiek materiałów ze strony jest surowo zabronione bez zezwolenia. [Warunki korzystania z witryny](https://www.sigmaaldrich.com/PL/pl/life-science/legal/site-use-terms)|[Polityka prywatności](https://www.sigmaaldrich.com/PL/pl/life-science/legal/privacy-statement)|[Ogólne warunki sprzedaży](https://www.sigmaaldrich.com/PL/pl/life-science/legal/terms-and-conditions)|[Zgoda na prawa autorskie](https://www.sigmaaldrich.com/PL/pl/life-science/legal/copyright-consent)|[Mapa witryny](https://www.sigmaaldrich.com/PL/pl/site-map)