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Staining

Nuclear & Cytoplasmic Staining

QuestionAnswer
Resinous mounting media Natural or synthetic resin, 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 bad for stain, add cane sugar to prevent dye bleeding, may need to seal coverglass w/ glue/nail polish, refractive index 1.41-1.43
Schiff Reagent solution sodium metabisulfite + HCl = sulfurous acid >> red. fuchsin basic fuchsin activated charcoal distilled water
Feulgen Reaction reagents Schiff reagent HCl sulfurous acid light green
Feulgen Reaction facts Fixative: any except Bouin QC: none, all nuclei give positive reaction, timing of hydrolysis critical
Fuelgen Reaction results DNA - reddish purple cytoplasm (if counterstain w/ aq. light green) - light green
Regressive Staining Tissue is overstained & excess stain removed until desired intensity (differentiation/decolorization)
Regressive Staining Results nuclei - blue rbc & eosinophilic granules - bright pink to red cytoplasm & other - various pinks
Which hematoxylin solutions are used regressively? Delafield, Ehrlich, Harris w/o acetic acid, be careful w/ differentiation
Progressive Staining Tissues in solution until desired intensity reached, highly selective, dye concentration & mordant choice more important
Progressive Staining Results nuclei - blue rbc & eosinophilic granules - bright pink to red cytoplasm & other - shades of pink
Which hematoxylin solutions are used progressively? Mayer, Harris w/ acetic acid
Eosin-Phloxine B solution eosin Y phloxine B acetic acid 95%
Eosin facts Usual H&E counterstain, 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 w/ same results, b/c mordant & oxidizer are same solution not stable for long
Gill Hematoxylin I solution aluminum sulfate sodium iodate ethylene glycol acetic acid anhydrous 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
Aluminum is the mordant in_ Harris, Delafield, Mayer, Ehrlich, Gill
Iron is the mordant in_ Weigert
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 distilled water Methyl Green Solution: acetate buffer methyl green dye
Methyl Green-Pyronin Y facts Fixative: 10% NBF; B5, Helly, Zenker ok, QC: formalin-fixed, paraffin w/ 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, distilled water Giemsa, glycerin, acetic water, 100% methanol, distilled water
May-Grunwald Giemsa facts Fixative: 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
Methyl Green-Pyronin Y purpose Differentiate between RNA & DNA, ID plasma cells or immunoblasts
Feulgen Reaction purpose Demo DNA
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 in compound, more color
Fundamental chromophore groups C=C, C=O, C=S, C=N, N=N, N=O, NO2
What is a chromagen? Benzene derivative w/ chromophores, colored, may or may not be able to act as dye
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 (potassium permanganate, chromium trioxide)
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 distilled water
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
Frozen H&E results Nuclei - blue Cytoplasm & other - various shades of pink
3 ways to restore tissue basophilia 1) if b/c Bouin - LiCO3 1 hr - wash & stain 2) sodium bicarbonate 3 hr (longer if b/c Zenker) - wash & stain 3) aq. periodic acid 30 min - wash x3 & stain
IEP of proteins? pH at which protein is neutral, 6/(7), pH of solution 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 those that_ Combine basic dye, e.g. methylene blue, w/ acidic dye, e.g. eosin
Polychromatic stains are_ Compound dyes/dye mixtures, e.g. Romanowsky stains, nuclear/cytoplasmic stains b/c range of cytoplasmic staining
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
Created by: CCF