Neutral red is a basic azine dye which is widely used in numerous staining techniques because of its affinity for chromatin [1,2] and its potential as a vital dye [3,4]. Although the fluorescence properties of neutral red are known [5], the use of this dye as a direct fluorochrome has been scarcely explored.The chemical structure of neutral red shows inter esting similarities with other planar cationic dyes belonging to the acridine, thiazine, and xanthene groups [2]. Like these dyes, neutral red [6] as well as other azine derivatives [7,8] were shown to interact with DNA. Basic planar dyes bind to nucleic acids by two different mechanisms, namely, an external interaction with phosphate groups, and the inter calative binding mode, in which the dye monomer is located between two base pairs [9][10][11]. The aim of this work is to describe the chromatin fluores cence and staining characteristics by neutral red in terms of binding mechanisms.Smears of chicken blood were fixed in methanol for 5 min and air dried. Staining was performed at room temperature by using neutral red (Merck, without further purification) solutions in distilled water, diluted from a 10~3 M stock solution. Staining time was 5 min after which slides were briefly washed in distilled water and air dried. Before Reprint request to Prof. Dr. Juan C. Stockert. 0341-0382/82/0100-0139 $ 0 1 .0 0 /0 staining, the following extraction procedures were applied: 5% trichloroacetic acid (TCA) at boiling temperature for 30 min; DNase I (Serva, 0.5 mg/ml in 1 mM MgCl2) and RNase A (Sigma, 1 mg/ml in distilled water), both used at room temperature for 2 h. Some stained smears were subjected to postreatments with 2% aqueous solutions of potassium alu minium sulfate and barium chloride for 3 min. In other cases, neutral red staining was carried out after treatment with methylene blue (Fluka). Obser vations were made in a Zeiss Photomicroscope III equipped with an epifluorescence condenser III RS and the filter set for green exciting light (546 nm). Cytofluorometric and cytophotometric measure ments were carried out according to the previously described procedures [12,13].At high concentration (> 1 0~*m) neutral red stained the chromatin from erythrocyte nuclei in a deep red color. On the contrary, smears subjected to lower dye concentrations (<10~4m) showed un stained nuclei under bright field illumination but an intensively red chromatin fluorescence. Extraction procedures such as TCA and DNase abolished the staining and fluorescence reaction of nuclei by neutral red, while treatment with RNase was with out effect. These results indicate that DNA is the chromatin component responsible for dye binding.The intensity of chromatin staining and fluores cence after teatment with different concentrations of neutral red is shown in Fig. 1 showed an increasing red emission, meanwhile nuclei appeared with a decreasing fluorescence. It is already known that the emission characteristics of fluorochromes are strongly dependent on the con centration and the type of binding m...

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Hematoxylin, Fluorescence, Fluorochrom es Treatments o f chicken blood and Ehrlich ascites tumor smears with hematoxylin solutions give a fluorescence reaction in chromatin, basophilic cytoplasm and leukocyte granules. In these structures the fluorescence emission in­ creases upon dye aging and prolonged staining times. We present a preliminar spectral analysis suggesting the possibility to employ hematoxylin as a fluorochrome.

We studied the effectiveness of harmine as a substitute for 33258 Hoechst in the fluorescence-plus-Giemsa technique, using Allium cepa chromosomes after 5-bromo-2'-deoxyuridine (BrdU) incorporation. Harmine showed a photosensitizing capacity which was somewhat higher than 33258 Hoechst and used half of the time established for the usual treatment.