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CR Sheue 03/ 2018 • Basic • Acid Dye CR Sheue 03/ 2018 • Basic • Acid • Natural dye • Synthetic Safranin O 番紅 and Fast-green • Safranin: basic dye, lignin, chromosome, nucleolus and cuticle red • Fast-green: acid, other part of cells green • Commonly used for plant organs, especially for mature tissue. • Counterstain (對比染色) of Safranin O and fast-green sols.unlv.edu/.../CellsTissues/Cells.html Bamboo vascular bundles. (Left to right): light micrograph with safranin/alcian blue stain. http://www.steve.gb.com/science/electron_microscopy.html Safranin O - Knee Joint bone.mcgill.ca/histo.gallery.pics.php Pollen of Petunia grandiflora, Pollen grains of Ipomoea colored idem (同上). with Fast Green and mounted in PVA-G, Obj. x 100. http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artmay04/wdstains.html Figure 5: In vivo demonstation of sustained microinfusion of Fast Green. (A) Twelve hours after intrastriatal placement of a Fast Green-loaded microinfusion system, dye is seen diffusing into the parenchyma surrounding the microcannula site. Observations at 1 day (B), 2 days (C), and 4 days (D) indicate continued delivery of Fast Green solution with increasing areas of dye infiltration. The outer limit of diffusion was observed under low-power microscopy and is indicated here with dashed lines. Scale bar, 2 mm. www.jove.com/index/details.stp?ID=716 JOHANSEN’S SAFRANIN AND FAST GREEN • Johansen’s method (Johansen, 1940) differs from most other Safranin/Fast Green protocols in the use of additions to the stain and clearing solutions to enhance and differentiate tissue structure. (See the staining schedule for notes on the purpose of the additions.) It is widely agreed that this method yields a more brilliant staining of plant tissues than almost any other schedule. Safranin O is a regressive dye and requires destaining and differentiation with picric acid or HCl. The Safranin-stained tissues are counterstained with Fast Green — a progressive dye. At the concentration used, Fast Green stains the tissues within 10–15 s, so it is best to test the procedure on one or two slides before staining your entire set. • In plant tissues stained with this method, Safranin O appears brilliant red in chromosomes, nuclei, lignified, suberized, or cutinized cell walls. Fast Green appears a brilliant green in cytoplasm and cellulosic cell walls. Fast Green turns blue in basic solutions. It appears blue to bluish-green in the stems and leaves of aquatic plants and most gymnosperms (Johansen, 1940). An aqueous alternative to Fast Green FCF is Alcian Blue. http://microscopy.berkeley.edu/Resources/instruction/staining.htm Haematoxylin (hematoxylin) 蘇木精 • Natural dye, from wood of Haematoxylon campechianum • Oxidation -- hematein (mature) + mordant • Types: Delafield, Heidehain, … Haematoxylon campechianum http://www.csdl.tamu.edu/FLORA/imaxxfab.htm 墨水樹 Loss-of-function mutations in the rice homeobox gene OSH15 affect the architecture of internodes resulting in dwarf plants http://www.nature.com/emboj/journal/v18/n4/fig_tab/7591533ft.html Fig. 6.Sections of the fourth internodes of wild-type and d6 dwarf mutant plants. (A) Longitudinal section through the IM of the fourth internode of a wild-type plant. Bar = 100 m. (B) Longitudinal section through the IM of the fourth internode of a d6 mutant plant. Bar = 100 m. (C) Longitudinal section through the elongated zone of the fourth internode of a wild-type plant. Bar = 100 m. (D) Longitudinal section through the elongated zone of the fourth internode of a d6 mutant plant. Bar = 100 m. (E) Cross sections of the fourth internodes of wild-type. Bar = 100 m. (F) Cross sections of the fourth internodes of d6. Bar = 100 m. (G) Higher magnification of (E). Bar = 30 m. (H) Higher magnifications of (F). Bar = 30 m. Note that sections in panel (A) and (C) were stained with hematoxylin and the rest of sections were stained with safranin. ep, epidermis; sc, sclerenchymatous cell layers; sv, small vascular bundles; lv, large vascular bundles. (hematoxylin-eosin, × 200): Type AB thymoma. Note type A and type B components. (hematoxylin-eosin, × 200) www.diagnosticpathology.org/.../2/1/13/figure/F3 Aniline blue 苯胺蓝；中國藍；青瓷色 • Acidic, algae, especially whole mount (胼胝質) Callose is a plant polysaccharide. It • Callose is composed of glucose residues linked together through β-1,3- linkages, and is termed a β-glucan. It is laid down at plasmodesmata, at the cell plate during cytokinesis and during pollen development. Water blue Case 1 • In order to demonstrate Callose in fresh material we will use free hand longitudinal sections of Cucurbita stained with Aniline Blue. • Aniline Blue preferentially stains callose. • Furthermore, stained callose emits fluorescence under ultra-violet and violet light. http://www.biologie.uni-hamburg.de/b-online/library/webb/BOT410/410Labs/LabsHTML-99/Xylem/Labxyphlo99.html Detailed protocol for Aniline Blue Staining • Place sections in IKI for 3 minutes, • Rinse with water • Stain 5 minutes with 0.1% aqueous aniline blue. • Wash briefly with IKI • Mount in water. http://www.biologie.uni-hamburg.de/b-online/library/webb/BOT410/410Labs/LabsHTML-99/Xylem/Labxyphlo99.html Overall view of a longitudinal section of cucumber stem stained with Aniline Blue and seen with Violet Fluorescence. The cell walls of the Xylem are auto fluorescent while the fluorescence of the Phloem is due to Callose which has stained with Aniline Blue. The sieve plates will be the most fluorescent areas because callose accumulates there normally and becomes more concentrated after wounding. http://www.biologie.uni-hamburg.de/b-online/library/webb/BOT410/410Labs/LabsHTML-99/Xylem/Labxyphlo99.html Case 2 Pollen tube from EXPB1 pollen growing through ovary tissue for 22 h after pollination. Ovaries were stained with 0.1% aniline blue for 30 min and then examined under a fluorescence microscope http://www.plosone.org/article/slideshow.action?uri=info:doi/10.1371/journal.pone.0000154&imageURI=info:doi/10.1371/j ournal.pone.0000154.g004 Aniline Blue Staining of Pollen/Pollen Tubes 花粉管染色 • Fixative: 10% Acetic Acid in ethanol 1M NaOH 50 mM KPO4 buffer, pH 7.5: 4.17 mL 1M K2HPO4 0.83 mL 1M KH2PO4 0.01% aniline blue in 50 mM KPO4 buffer (dye) KPO4 buffer made with 50% glycerol (mounting media) Method 1. Submerge pistil tissue in about 250 µL acetic acid fix for 1.5 hrs or more. An eppendorf tube works well for this. Tissue can be left in fix overnight or longer if necessary. 2. Soften tissue by submerging tissue in 1 M NaOH overnight. 3. Wash 3 times with 50 mM KPO4 buffer. Be very gentle with the tissue because it is fragile at this stage. 4. Stain with 200 µL aniline blue for 5-10 minutes. 5. Transfer to slide, add mounting media, and observe under UV. Squash if necessary. http://www.5ibio.com/html/jibenshiyanjishu/dongzhiwu/20070122/2774.html Material suggested • Oxalis corymbosa 鹽酸仔菜、酸味草、黃花酢漿草 Carmine • Basic, from Coccus cacti (insect) in tropical Amer. • Algae, bacterial, chromosome • Applied with smear method Female (left) and male (right) Coccus cacti Cochineals • Carmine also called Crimson Lake, Cochineal, Natural Red 4, C.I. 75470, or E120, is a pigment of a bright red color obtained from the carminic acid produced by some scale insects, such as the cochineal and the Polish cochineal, and is used as a general term for a particularly deep red color. Carmine is used in the manufacture of artificial flowers, paints, rouge, yogurt, cosmetics, food additives, and crimson ink. Orcein • Orcein, also archil, orchil, lacmus, litmus, Citrus Red 2, and C.I. Natural Red 28, are names for dyes extracted from several species of lichen, also called orchella weeds, found in various parts of the world. Commercial archil is either a powder (called cudbear) or a paste. It is red in acidic pH and blue in alkaline pH. • Orcein is approved as a food dye, with E number E121. Its CAS number is [1400-62-0]. Its chemical formula is C28H24N2O7. It forms dark brown crystals. • Can be used to stain elastic fibers found in connective tissue. http://en.wikipedia.org/wiki/Cudbear • Orcinol is extracted from archil lichen, . It is then converted to orcein by ammonia and air. Orcein is a reddish-brown dye, orchil is a purple-blue dye. Orcein is also used as a stain in microscopy to visualize elastic fibers. It is a mixture of derivates - hydroxyorceins, aminoorceins, and aminoorceinimines. TBO • Toludine Blue O is another basic dye. It's used as a quick stain for light microscopic "orientation sections" used by electron microscopists. • Because it's so often used nowadays, more and more journal articles have images made from TB- stained preparations, so it's important to be able to interpret these images as readily as the more common ones made with H&E. The TB stain shows general structure in the same way as H&E does, and you can directly compare these two sections, which are approximately at the same magnification, find many features in common. Like H&E, TB provides only minimal information about the chemical makeup of a tissue or organ. Mast cell, toluidine blue • TB is also used clinically. It has a strong affinity for the granules in mast cells, one of the wandering cells of connective tissue. TB is often requested as a specific stain for mast cell tumors (肥大細胞腫瘤 mastocytoma). Transverse section through a leaf of Begonia formosana, stained with Toluidine blue (TB). Transverse section through a leaf of Phebalium glandulosum, stained with Toluidine blue. Transverse section of a prop root, stained with Toluidine blue. .
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