Technical Tips Online, Vol. 2, 1997

There is still life after death for‘Fast Red’ tabletsSabine Herblota, Farid Najemea, Catherine Lemoineb and Jacques Bonneta

aLaboratoire d’Immunologie Moleculaire, Zone Nord Bat 1b, Universite De Bordeaux II, 146, Rue Leo Saignat, 33076 Bordeaux Cedex,FrancebLaboratoire d’Hystologie-Embryologie, Zone Nord Bat 3b, Universite De Bordeaux II, 146, Rue Leo Saignat, 33076 Bordeaux Cedex,France

Keywords: Microscopy

▼Fast Red (Boehringer Mannheim), a dye used as a col-orimetric substrate for alkaline phosphatase, is rather shortlived. Indeed, at 4◦C, the tablets can be stored for only 6months and solutions have to be used within 30 min. How-ever, out-of-date solutions and tablets can still be of use;one of us (S.H.) realized that one of the degradation prod-ucts is a sensitive fluorimetric substrate of alkaline phos-phatase. Indeed, when Fast Red tablets more than one yearold and solutions more than 15 days old are used withdigoxigenin-labeled DNA following the supplier’s protocol,no staining is seen with visible light. However, a fluores-cent insoluble product has been formed (Fig. 1). The flu-orimetric substrate seems to be fairly stable and the sen-sitivity of detection of digoxigenin-labeled DNA actuallyincreases after storage of the solution at 4◦C for up to 2

Corresponding author: jacques.bonnet@immol.u-bordeaux2.fr

weeks. The use of this substrate for in situhybridization wasalso tested, and results compared with those obtained withNBT-BCIP (4-nitro blue tetrazolium chloride−5-bromo-4-chloro-3-indolyl phosphate, Fig. 2).

References1 Julien, J.F., Samama, P. and Mallet, J. (1990) J. Neurochem. 54, 703–705.2 Moine, C., Normand, E. and Bloch, B. (1995) Cell. Mol. Biol. 41, 917–923.

Products UsedFast Red dye: Fast Red dye from BoehringerMannheim

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Technical Tips Online, Vol. 2, 1997 Technical Tips

FIGURE 1. Digoxigenin-labeled DNA was serially diluted, dotted on a nylon membrane and UV-cross-linked. The blot was treated as described by thesupplier of Fast Red. The blot was illuminated with UV light at 254 nm and photographed with a camera coupled with NIH image processing software(inset). A, Dot blot processed with a fresh solution of Fast Red prepared with 12-month-old Fast Red tablets; B, dot blot processed 15 days later with thesame solution.

FIGURE 2. Insituhybridization of digoxigenin-labeled glutamic acid decarboxylase (GAD; Ref. 1) probe on brain tissue (striatum). Insitu hybridization wasconducted as described previously (Ref. 2). Fluorescent detection with two-year-old Fast Red at 546 nm (a) gives a similar result as with NBT-BCIPdetection (b). The labeled cells are γ -butyric acid (GABA)-positive neurons, imbedded in negative glial cells and a few (5%) GABA-negative neurons.

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