Orcein stain

Allow 10 min for fixation. The coverslip is then allowed to stand for 5 min or until absolutely dry. Stain for 10 min with orcein stain (Appendix 2) or longer if necessary and wash 3 times in absolute alcohol. 

Clausen PP, Thomsen P. Demonstration of hepatitis B surface antigen in liver biopsies. A comparative investigation of immu-noperoxidase and orcein staining on identical sections on formalin-fixed, paraffin-embedded tissue. Acta Pathol Microbiol ScandfA]. 1978 86A 383. 

Fig. 10.6.7a,b. Transvascular migration of non-spherical PVA particles and tris-acryl microspheres bilateral uterine artery embolization in the sheep was performed 30 months earlier (orcein staining), a An aggregate of PVA particles (in black) is located in the media. No PVA particle is seen in the media or outside of the vessel. The internal elastic limitans is ruptured. A recanalization of the embolized artery is visible, b One tris-acryl microsphere (in white) is visible outside the vessel. Both internal and external elastic limitans are ruptured and indicate the trajectory of the microsphere exclusion. A recanalization of the embolized artery is also visible... 

Mori, J. Omata, M. Yokosuka, O. Imazeki, F. Ito, Y Uchiumi, K. Matsuyama, Y. Ye, W. F Okuda, K. Liver orcein stain and viral DNA in duck hepatitis B virus infection in Chinese ducks and experimentally infected Japanese ducklings. Hepatology 1984, 4, 1124-1128. 

Panicker, J. N. Shenoy, K. T. Augustine, J. A cytological study of hepatitis B surface antigen localization using orcein staining in hepatocellular carcinoma. Indian J. Med. Res. 1996,104, 374—376. 

Murthy, D. P SenGupta, S. K. Kelkar, S. S. Orcein staining for hepatitis B surface antigen (HBsAg) in liver diseases in Papua New Guinea. Papua New Guinea Med. J. 1988, 31, 179-183. 

PREPARATION AND STAINING OF LARVAL BRAIN MITOTIC CHROMOSOMES, 3 Preparation of Orcein-stained Chromosomes, 4 Preparation of Unstained Chromosomes, 4 Giemsa Staining, 4... 

Several techniques have been developed for preparation of male meiotic chromosomes (see, e.g.. Cooper 1965 Ashbumer 1989 Miyazaki and Orr-Weaver 1992). All of these techniques allow a clear visualization of chromosomes, but not of other cellular structures such as the cytoskeleton and the spindle. We describe here three of these techniques that in our hands give good reproducible results. The first technique, developed by Lifschytz and Hareven (1977) and described below in Protocol 5.9, is particularly usefid for the analysis of adult testes. The other two techniques (Ripoll et al. 1985 Cenci et al. 1997) are routinely used in our laboratory for larval and pupal testis preparations. The technique described in Protocol 5.10 is a very simple method for preparing aceto-orcein-stained chromosomes. The other technique outlined in Protocol 5.11 allows preparation of unstained chromosomes that can be subsequently stained with various dyes such as Giemsa, Hoechst 33258, and DAPl, or processed for in situ hybridization. 

Heat the staining solution (but do not boil) and filter. If stored tightly capped, it will last for many years. The same solution, but without the orcein stain, can be used to prepare polytene chromosomes for in situ hybridization. 

Staining of cells may be done before or after the autoradiography and Giemsa (Appendix 2) is generally recommended. Some stains, e.g. aceto-orcein will remove silver grains and have to be used before covering the cells with emulsion. 

Mention should be made of oxazine dyes, used also as biological stains, which are oxidized phenoxazine derivatives containing suitable auxochromic groups. A detailed treatment of these dyes, however, is beyond the scope of this review. Most of the industrial phenoxazine dyes are derived from benzophenoxazines (e.g., Meldola s blue) or from more complex ring systems containing the phenoxazine residue (triphendioxazine dyes).3,118 The long-known dyestuffs orcein and litmus which are prepared by the action of ammonia on certain lichens, and may also occur accidentally in nature, are both based on the oxidized phenoxazine ring system as shown by Musso and co-workers.119... 

Use Mainly in microscopy to stain elastic fibers, acidic mucines, cartilage, etc. The names orcein, orcinol, and Orseille are purported to originate via Oicela from the name of the Florentine merchant family Ro-cela who possessed a monopoly for lichen dyes in the 14th century. 

Stain slides with 2% lactic-acetic-orcein by adding a drop of stain to the slide and then placing a cover glass onto it. After some 2 min, gently press the cover glass and the slide within a bibulous pad to remove excess of fluid. Slides prepared this way do not dry up for at least a month. 

Dissolve 2 g of orcein (natural) in 45 ml of hot glacial acetic acid. When the solution is cooled, add 7.5 ml of distilled water and 47.5 ml of lactic acid. Filter and store (4°C) in a brown bottle. Filter the stain periodically. Long storage will result in precipitation of the stain. 

Staining procedure When dry, invert into a drop of 1.0% acetic orcein (0.5 g orcein in 45 ml glacial acetic acid reflux % hr, add 55 ml distilled water while warm, reflux % hr, let stand 24 hr, filter, and filter fresh before using) on a clean slide, suck out excess out excess stain with filter paper, and seal with Kroenig s cement. If permanent slides are desired treat dry coverslip preparation in following sequence 1.0% acetic orcein (30 min), 45% acetic acid (dip until free of excess stain), tertiary butyl alcohol equal equal xylene (1 min), xylene (1 min), xylene (1 min), and invert wet into Permount (thinned with xylene) on clean slide. 

Staining can be achieved by standard procedures utilizing acetic-orcein, Giemsa stain, or the Feulgen reaction. 

Staining may be achieved by acetic-orcein, lactic-acetic-orcein, or 2% toluidine blue. 

Small pieces of tubules (1-2 cm length) are placed on a slide and stained with a drop of lactic-acetic-orcein. 

The preparations can be stored or stained immediately with lactic-acetic-orcein, toluidine, or Unna blue. Toluidine blue is generally preferred for immediate examination of nonpermanent preparations. Lactic-acetic-orcein and Unna blue are used for finer examination and for permanent preparations. 

Fig, 51, a) Microautoradiograph of two chromosomes from salivary gland nuclei of Drosophila after incorporation of -thymidine (15 pCi/ml, 10,000 juCi/pM) for 5-8 min b) phase contrast of the same field after staining with aceto-orcein and before application of autoradiographic emulsion (x 1900). Absence of absolute correlation between density of bands and presence or absence of label will be noted (Plant and Nash, 1964). 

Lavania, U. C. Sharma, A. K. Trypsin-orcein banding in plant chromosomes. Stain Technol. 1979, 54, 261-263. 

Goldstein, D. J. Selective staining of eosinophil granules in sections by alkaline orcein in a concentrated urea solution. Stain Technol. 1963, 38, 49-51. 

El-Maghraby, M. A. H. A. Gardner, D. L. Synthetic orcein as a stain for chick embryo cartilage matrix. Stain Technol. 1969, 44, 127-129. 

Kimoto, H. Oda, T. Detection of histamine in rat mast cell granules by orcein-water blue stain. Acta Histochem. Cytochem. 1979, 12, 292-300. 

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