Biotin-labeled DNA probes, prepared by nicktranslation in the presence of biotinylated analogs of TTP, are hybridized to DNA or RNA immobilized on nitrocellulose filters. After removal of residual probe, the filters are incubated for-2-5 min with a preformed complex made with avidin-DH (or streptavidin) and biotinylated polymers of intestinal alkaline phosphatase. The filters are then incubated with a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium, which results in the deposition of a purple precipitate at the sites of hybridization. This procedure will detect target sequences in the 1-to 10-pg range after enzyme incubation periodwof 1 hr or less. The incubation period can be extended up to 24 hr, if required, to increase the color intensity of the hybridization signal. Furthermore, at high probe concentrations (250-750 ng/ml), biotin-labeled DNA exhibits lower nonspecific binding to nitrocellulose than does radiolabeled DNA, so hybridization times required for the analysis of unique mammalian gene sequences can be decreased to 1-2 hr. This nonradiographic method of probe detection should be of general utility for genetic studies using Southern, RNA, or dot-blot hybridization protocols.Previous reports from this laboratory described the synthesis of biotin-labeled analogs of TTP and UTP that can be enzymatically incorporated into DNA and RNA, respectively (1, 2). The resulting biotin-labeled polynucleotides exhibit reassociation kinetics similar to those of biotin-free polymers and they function effectively as hybridization probes in situ. Hybridization signals can be visualized by indirect immunofluorescence, immunoperoxidase, or immuno-colloidal gold techniques, after incubation with a primary antibiotin antibody, and by cytochemical methods that use complexes of avidin and biotinylated peroxidase to detect the biotin-labeled probe. Such procedures have been applied successfully to the localization of specific sequences in Drosophila chromosomes (3, 4), mammalian metaphase chromosomes (5, 6), cultured cells (2, 7), and formalin-fixed tissue sections (2).However, none of the visualization methods used in these studies were able to detect sequences present at the level of one copy per mammalian cell. It was apparent, therefore, that the routine application of biotin-labeled probes in genetic analysis would require the development of more sensitive biotindetection systems.Here we report the synthesis of biotinylated polymers of alkaline phosphatase and the construction of complexes of avidin (or streptavidin) and enzyme polymer that are 20-to 50-fold more sensitive than immunologic or affinity reagents used previously. We also describe a rapid and sensitive procedure for visualizing biotin-labeled DNA probes after hybridization to DNA or RNA immobilized on nitrocellulose filters.

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Purified bovine brain calmodulin was biotinylated with biotinyl-e-aminocaproic acid N-hydroxysuccinimide. Biotinylated calmodulin was used to detect and quantify calmodulin-binding proteins following both protein blotting and slot-blot procedures by using alkaline phosphatase or peroxidase coupled to avidin. When purified bovine brain calcineurin, a calmodulin-dependent protein phosphatase, was immobilized on nitrocellulose slot blots, biotinylated calmodulin bound in a calcium-dependent saturable manner; these blots were then quantified by densitometry. Biotinylated calmodulin was able to detect as little as 10 ng of calcineurin, and the binding was competitively inhibited by addition of either native calmodulin or trifluoperazine. When biotinylated calmodulin was used to probe protein blots of crude brain cytosol and membrane preparations after gel electrophoresis, only protein bands characteristic of known calmodulin-binding proteins (i.e., calmodulin-dependent protein kinase, calcineurin, spectrin) were detected with avidin-peroxidase or avidinalkaline phosphatase procedures. Purified calcineurin was subjected to one-and two-dimensional gel electrophoresis and protein blotting; as expected, only the 61-kDa calmodulinbinding subunit was detected. When the two-dimensional protein blot was incubated with biotinylated calmodulin and detected with avidin-alkaline phosphatase, several apparent forms of the 61-kDa catalytic subunit were detected, consistent with isozymic species of the enzyme. The results of these studies suggest that biotinylated calmodulin can be used as a simple, sensitive, and quantifiable probe for the study of calmodulinbinding proteins.Much of our current understanding of calmodulin-dependent control of cellular functions has stemmed from identification of the calmodulin-binding proteins, which are activated by Ca2+-calmodulin (1). Calmodulin has been shown to mediate activation of a number of calcium-dependent enzymes such as phosphodiesterase (2-4), adenylate cyclase (5), calmodulin-dependent protein kinases (6)(7)(8) (14) to immobilize proteins on nitrocellulose paper followed by incubation of the Tween-20 blocked paper with 1251I-labeled calmodulin (15).Avidin-biotin probes have been used to visualize biotinlabeled DNA in nitrocellulose blot hybridization studies (16). We have exploited the high-affinity avidin-biotin interaction (17) to afford detection of biotinylated calmodulin and calmodulin-binding proteins. We now report that biotinylated calmodulin can effectively bind both purified and crude preparations of calmodulin-binding proteins with sensitivity of detection in the nanogram range. Biotinylated calmodulin has also been used to develop a quantitative slot-blot procedure for the quantification of individual calmodulin-binding proteins. We have also used this approach to probe twodimensional gels of purified calcineurin and have found several apparent (isozymic) forms of the 61-kDa catalytic subunit (18). The use of biotinylated binding proteins as probes and detection ...

We report the rapid (less than 1 hr), immunocytochemical identification of various fungi in formalin-fixed, paraffin-embedded tissues using antisera originally developed for use in immunodiffusion assays. Primary antisera directed towards fungal genera including Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Histoplasma, and Sporothrix were examined. The specificity of each antiserum was evaluated by the presence or absence of crossreactivity with other morphologically similar fungi in both paraffin-embedded pure fungal cultures and tissues with culture-confirmed fungal infections. Each antiserum reacted strongly with the fungus to which it had been raised, whether examined in pure culture or infected tissues. The antisera raised against Candida, Cryptococcus, and Sporothrix did not exhibit cross-reactivity with any other fungus tested. However, the antisera raised to Aspergillus, Blastomyces, Coccidioides, and Histoplasma demonstrated significant crossreactivity with other genera of fungi, thus precluding their routine use in diagnostic immunocytochemistry. The results indicate that immunocytochemistry may provide an important adjunct to other methods, such as immunodiffusion or complement fixation assays and histochemical stains such as the Grocott methenamine silver or periodic acid-Schiff, when attempts are made to specifically identify certain fungi in formalin-fixed, paraffin-embedded tissues before mycology culture results are available.

We describe the development of a rapid colorimetric in situ hybridization technique utilizing oligonucleotide probes labeled with six biotin molecules at the 3' end to detect mdrl in mouse colon cancer cells growing in culture and in vivo. mRNA integrity was vedied by the use of a multibiotinylated poly d(T) oligonucleotide, and the specificity of the reaction was confirmed by use of labeled sense and anti-sense probes in serial cryostat sections and cultured cells. The multiple biotin label produced a strong signal after a short hy-