HTL Nuc/cytop stains
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| define adsorption | bound chemically as opposed to "soaked up"
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| define absorbtion | "soaked up" contained within crevasses or spaces like water in a sponge
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| define mordant | substances, often metals, that act as a link between the dye and the tissue
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| define basophilia | base loving. Substances that attract/are attracted to basic dyes
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| define acidophilia | acid loving. Substances that attract/are attracted to acid dyes
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| define ripening | the oxidation of hematoxylin to the active dye: hematein
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| define auxochrome | an ionizing group required to enable dye to link to tissue
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| define chromophore | a group of atoms that confers the property of color onto a compound. they are all easily reduced, which destroys the color.
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| define polychrome | A compound dye that contains components of different colors
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| define cationic dye | A basic dye. One where the charge of the dye ion is positive. the auxochrome is the amino group NH2
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| define anionic dye | An acid dye. One where the charge of the dye ion is negative. Auxochromes are carboxyl COOH, hydroxyl OH, and sulfonic acid SO3H groups.
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| define dye lake | mordant combined with dye forms a lake
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| describe and differentiate heterochromatin and euchromatin | heterochromatin is the inactive form of chromatin which can be stained and visualized. euchromatin is activly involved in transcription and cannot be stained or seen without electron microscopy
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| how can cell activity affect H&E staining | active DNA will stain less intensely (more euchromatin) rough endoplasmic reticulum may also stain giving appearance of basophilic cytoplasm.
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| differentiate hematin and hematein | hematein is the oxidized dye in the logwood tree wood. hematin is formalin pigment.
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| define progressive and regressive staining | progressive: staining progress is monitored until desired color/intensity is achieved, then staining is stopped. regressive: tissue is over-stained and subsequently differentiated.
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| which hematoxylins are progressive stains? | Harris, Gill, Mayer
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| which hematoxylins are regressive | Delafield, Ehrlich, weigert
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| list 3 methods of differentiation | 1 acid/base differentiation. 2 exces mordant differentiation. 3 oxidative differentiation.
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| list 4 natural dyes and their sources | hematoxylin (heartwood of logwood tree), orcein (lichens), carmine (tropical cochineal insect), saffron (pistils of flowering plants)
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| describe Ehrlich hematoxylin | Mordant: aluminum. Oxidizer (ripening): natural or chemical. commonly regressive, can be used progressively.
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| describe Delafield hematoxylin | Mordant: aluminum. Oxidizer (ripening): natural. Stabilized with glycerol against overoxidation and evaporation. Intended as regressive stain.
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| describe Harris hematoxylin | Mordant: aluminum. Oxidizer (ripening): sodium iodate. Acidified with acetic acid for specificity. Intended as progressive stain.
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| describe Mayer hematoxylin | Mordant: aluminum. Oxidizer (ripening): sodium iodate. Stabilized against scum/precipitate and pH balanced with citric acid and chloral hydrate. Progressive stain. recommended for immunoperoxidase techniques with 3-amino-9-ethylcarbazole as chromogen.
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| describe Gill hematoxylin | Mordant: aluminum. Oxidizer (ripening): sodium iodate, recommended to ripen for 1 week @ 37C. Ethylene glycol provides excellent solvent for hematoxylin and prevents surface precipitates. Will stain mucin
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| describe Weigert hematoxylin | Mordant:Ferric chloride (iron). Oxidizer (ripening): Ferric chloride. Used in non-routine applications as it resists decolorization in acidic solutions (eg trichrome)
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| why would one use Iron hematoxylin? | To resist decolorization in acidic solutions
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| how would excess acid or aluminum effect hematoxylin staining | more selective nuclear staining can be achieved.
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| how does pH effects H&E staining? | pH determines whether a dye will bind to tissue elements by establishing the appropriate charges on both the tissue and dye molecule.
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| list three factors other than pH that affect staining | temperature, dye concentration, salts other than the dye itself in solution
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| what happens to staining when tissues have been overexposed to acidic solutions | They will lose their basophilia (nuclear staining)
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| how do formalin and zenkers affect staining | Zenkers is acidic and overexposure can inhibit basophilia of nuclei. Formalin takes up amino groups that normally bind eosin and can increase cytoplasmic basophilia
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| ID 6 sources of error in H&E | water in processing reagents, over/under oxidized hematoxylin, improper differentiation/bluing of eosin/HT, loss of nuclear basophilia from fixation, caryover of bluing soln. to eosin removes staining etc.
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| ID 2 types of nucleic acids and 2 methods to stain them | DNA RNA. Hematoxylin, feulgen reaction, pyronin Y
(staining done with cationic/+charged/basic dye)
(staining done with dyes combined with metal mordants)
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| what specific substances are stained in nuclear stains | Fuelgen reactions remove purine bases and hydrolyzes pyrimidines to create a reactive aldehyde, then visualized with Schiff. Hematoxylin is believed to bind the phosphate groups of nucleic acids, but the reaction is not understood completely.
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| describe acid hydrolysis and fixation for Feulgen reactions | acid hydrolyzes the deoxyribose sugar in DNA to an aldehyde for visualization with Schiff reagent. Gluteraldehyde and picric acid fixation are not ideal as the former leaves aldehydes in tissue and the latter will over hydrolyze nuclei during fixation
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| list three dyes for nuclear staining | Hematoxylin, celestine blue, nuclear-fast red, methylene blue, thionin, toluidine blue O
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| describe the romanowski stains and their two uses | (Wright and Giemsa stains) Used for microorganisms and differentiating cells in hematopoietic tissues.
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| what are 2 classes of mounting media | Aqueous and resinous
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| give advantages of each media | Resinous media is permanent, dries quickly, has refractive index very near to tissue, and is compatible with most stains. Aqueous media will preserve stains that are destroyed or altered by dehydrating and clearing required for resinous media.
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| how and why does one seal coverslip edges | Nail polish, glue or other cement. this will preserve the aqueous mounting media against evaporation, smearing, etc. so that the slide may be stored in a more permanent fashion.
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| natural vs synthetic resinous mounting media | Natural resins are inherently acidic and will cause fading of some stains. They also tend to yellow with age, and may take days or months to set completely. Synthetic resins solve all of these problems.
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| types and thickness of coverslips and slides | the thicker the coverslip the less transparent the section will be.
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| refractive indexes of mounting media | Resinous= 1.51-1.55
Aqueous= 1.41-1.43
Tissue= ~1.53-1.54
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| coverslipping artifacts | media on slide, water under coverslip, dried sections prior to coverslipping, retracted media due to solvent dilution in the media.
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| 3 mounting problems | Cornflaking (sections dried before mounting) Water droplets (incomplete dehydration) retracted media (too thin at application)
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| Define amphoteric | substances that can carry both positive and negative charges depending on the pH.
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| What are the acid auxochromes? | COOH, OH, SO3H
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| What is a common basic auxochrome? | NH2
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| define adjective staining | a stain involving a substance that attaches the dye to the tissue, e.g. mordanting of hematoxylin
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| define substantive staining | a stain where the dye acts directly with the tissue, and does not require another substance
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| define impregnation | attaching a particulate metal salt onto or around a tissue element. It differs from traditional staining in that it does not penetrate the tissue
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| what is the purpose of the fuelgen reaction? | to demonstrate nuclei via hydrolysis of sugars to aldehydes and PAS
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| what are the reagents used in fuelgen reaction? | HCl --> DIH2O --> Shiffs --> water --> Sulfurous acid --> light green (option)
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| what is the purpose of the methyl green pyronin Y reaction? | demonstration of DNA and RNA, respectively
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| what are the reagents used in methyl green pyronin Y reaction? | methyl green (acetic acid, sodium acetate, mg)+pyronin Y to stain, acetone, xylene
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