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Nuclear & Cytoplasm
Staining
Question | Answer |
---|---|
Resinous mounting media | Natural or synthetic, cause fading of blue of Romanowsky, refractive index 1.5-1.55 |
Aqueous mounting media | Simple syrups, gum arabica, glycerol gelatins, use if dehydration & clearing harmful, add cane sugar to prevent dye bleeding, may seal coverglass w/ glue/nail polish, refractive index 1.41-1.43 |
Schiff Reagent solution | sodium metabisulfite + HCl = sulfurous acid + basic fuchsin + activated charcoal + dH2O |
Feulgen Reaction reagents | Schiff reagent + HCl (hydrolyzes to aldehydes) + sulfurous acid + light green |
Feulgen Reaction facts | Demo DNA, any fixative except Bouin QC: all nuclei give positive reaction, timing of hydrolysis critical |
Fuelgen Reaction results | DNA: reddish purple - cytoplasm if counterstained: light green |
Regressive Staining | Tissue is overstained & excess stain removed until desired intensity (differentiation/decolorization), Delafield, Ehrlich, Harris w/o acetic acid |
Progressive Staining | Tissues in solution until desired intensity reached, highly selective, dye concentration & mordant choice more important, Mayer, Harris w/ acetic acid |
Eosin-Phloxine B solution | eosin Y + phloxine B + acetic acid + 95% |
Eosin facts | Plasma stain, anionic, combine w/ amino acids, autofluoresces, best @ pH 4.6 to 5 |
Eosin-Phloxine B facts | more vivid pinks than eosin , a little orange, easy to overstain |
Eosin solution | eosin Y + acetic acid + 95% |
Celestine blue solution | celestine blue + ferric ammonium sulfate + dH2O |
Celestine blue fact | Can substitute gallein for celestine blue in iron hematoxylin methods |
Weigert Hematoxylin solution | ferric chloride + HCl + 95% + hematoxylin + dH2O |
Weigert Hematoxylin facts | Progressive 5-30 min, can substitute celestine blue for hematoxylin, not stable for long b/c mordant & oxidizer are same |
Gill Hematoxylin I solution | aluminum sulfate + sodium iodate + ethylene glycol + acetic acid + anhyd. hematoxylin + dH2O |
Gill Hematoxylin I facts | Better used progressively, only hematoxylin that stains goblet cells |
Harris Hematoxylin solution | ammonium aluminum sulfate + mercuric oxide or sodium iodate + 100% + hematoxylin + dH2O |
Harris Hematoxylin facts | Progressive, 1 to 3 min, acidify to pH 1.0 to 1.2 for very selective nuclear staining |
Mercuric chloride is the oxidizer in_ | Harris |
Light/air is the oxidizer in_ | Delafield, Ehrlich (aged) |
Sodium iodate is the oxidizer in_ | Harris, Mayer, Ehrlich (immediate use), Gill |
Mayer Hematoxylin Reagents | ammonium or potassium aluminum sulfate + sodium iodate + citric acid + chloral hydrate + hematoxylin + dH2O |
Mayer Hematoxylin facts | Progressive, slower, difficult to overstain, good for immunoperoxidase techniques b/c no alcohol |
Ehrlich Hematoxylin solution | ammonium or potassium aluminum sulfate + acetic acid + glycerol + 95% + hematoxylin + dH2O |
Ehrlich Hematoxylin facts | Usually regressive, can use progressive, ripen naturally or chemically |
Delafield Hematoxylin solution | ammonium aluminum sulfate + dH2O + glycerol + 95% + hematoxylin age |
Methyl Green-Pyronin Y solution | purified methyl green soln. + pyronin Y |
Methyl Green solution | Acetate buffer: sodium acetate trihydrate + acetic acid + dH2O Methyl Green Solution: acetate buffer + methyl green dye |
Methyl Green-Pyronin Y facts | To differentiate RNA & DNA, ID plasma cells or immunoblasts 10% NBF; B5, Helly, Zenker ok, QC: many plasma cells |
Methyl Green-Pyronin Y results | Immunoblast & plasma cell cytoplasm - intense red RNA - red Nuclei, DNA - green to blue green Goblet cells - mint green Background - pale pink to colorless |
May-Grunwald Giemsa solution | Jenner dye +methanol + dH2O Giemsa + glycerin +acetic water + 100% methanol + dH2O |
May-Grunwald Giemsa facts | Zenker, B5; 10% NBF ok QC: spleen pH: 6.4 to 6.9 |
May-Grunwald Giemsa purpose | Differentiate cells in hematopoietic tissue, demo some microorganisms |
May-Grunwald Giemsa results | nuclei, bacteria: blue = leukocyte cytoplasm : pink, gray, blue depending on cell type & development |
What are the two mechanisms of nuclear staining? | 1) Staining w/ basic dyes (req. presence of RNA or DNA); 2) staining w/ dyes plus mordant. |
Basic dyes | Cationic, positive charge, e.g. hematoxylin, crystal violet, some basophilic cell components: DNA, rough ER, RNA (ribosomes) |
Acidic dyes | Anionic, negative charge, e.g. eosin, orange G, picric acid, some acidophilic cell components: cytoplasm, muscle, conn. tissue, rbc |
Mechanism of cytoplasmic staining? | Based on charges of proteins, or side chains of amino acids. |
What is a chromophore? | Group that confers property of color, more chromophores present, more color |
Fundamental chromophore groups | C=C, C=O, C=S, C=N, N=N, N=O, NO2 |
What is a chromogen? | Benzene derivative w/ chromophores, colored, may or may not be able to act as dye, IHC chromogens DAB, AEC, produce colored precipitate |
Lipid Stains | Nonionic compounds, insoluble in water, soluble some organic solvents, dissolve into tissue to color (physical staining) |
Factors affecting dye binding | 1) Solution pH 2) Solution temperature 3) Dye molecule concentration 4) Competition from non-dye salts 5) Fixative |
3 ways to differentiate | 1) Weak acid or alkaline solution, 2) Excess mordant, dissociate dye from tissue 3) Oxidizers |
Hematein | Weak anionic dye, oxidation product of hematoxylin, affinity for nuclei when w/ metal mordant |
Lake | Mordant-dye combination |
Refractive index of tissue? | 1.53 to 1.54 |
Sulfurous acid solution | Sodium metabisulfite + HCl + dH2O |
Mordant | Metal or salt that forms complex w/ dye & attaches it to tissue |
Bluing | Scott's tap water, LiCo3, NH4OH, alkaline |
Clarifier | Acid alcohol, hematoxylin differentiator |
What hematoxylin is used for frozen H&Es? | Harris w/ acetic acid |
What type of mounting media should be used for frozen H&Es? | Synthetic resin |
3 ways to restore tissue basophilia | 1) if b/c Bouin - LiCO3, wash & stain 2) sodium bicarbonate (longer if b/c Zenker), wash & stain 3) aq. periodic acid, wash x3 & stain |
IEP of proteins? | pH at which protein is neutral, 6/(7) soln. pH determines charge & affects staining |
What is an auxochrome? | An ionizing group that enables dye to link to tissue, common auxochromes: -NH2, -COOH |
Most common histopath Romanowsky stain? | Giemsa |
Romanowsky-type stains are __ | Polychromatic stains that combine basic dye w/ acidic dye, as stain ages other dyes are formed esp. in alkaline pH |
Polychromatic stains are_ | Contains stains of several colors each of which stain selectively, Giemsa, May-Grunwald Giemsa, Wright's stain |
Nuclear membrane is stained by? | Aluminum hematoxylin solutions |
Chromatin includes_ | Stainable heterochromatin and nonstainable euchromatin, in the nucleus, basophilic |
Ribosomes are_ | Sites of protein synthesis, free ribosomes cause diffuse cytoplasmic basophilia, rough ER cause localized cytoplasmic basophilia |
Organelles that do not stain/are not seen in light H&E sections include: | Nuclear pores, plasmalemma, mitochondria, Golgi apparatus, centrioles |
Most commercial l Mayer hematoxylin solutions adjust pH w/_ | Acetic acid instead of citric acid |
Natural resin | Acidic, fade stains, Canada balsam, gum dammar, dissolved in xylene |
Synthetic resin | Neutral, permount, crystal mount, dissolved in toluene which is more reactive, more bubbles, dry quickly |
Amphoteric means: | **Substance can be positive or negative depending on pH of solution, some proteins & dyes are amphoteric |
Metachromatic means: | Tissue stained isn't same color as the dye, e.g. T blue stains mast cells pink |
Orthochromatic means: | Tissue stains same color as dye, e.g. light green CF stains tissue light green |
Common oxidation agents: | Atmospheric oxygen, sodium iodate, mercuric chloride, potassium permanganate |
Common mordants: | Ammonium aluminum sulfate, potassium aluminum sulfate, phosphotungstic acid, phosphomolybdic acid |
Scott's tap water solution | magnesium sulfate + sodium bicarbonate + tap water |
Some hematoxylin substitutes are: | Celestine blue, safranin, nuclear fast red, methylene blue, thionin, T blue O |
Common basic auxochrome: | -NH2 |
Common acidic auxochrome: | -COOH, the acidic auxochrome of eosin |