Chapter 6: Nuclear and Cytoplasmic Staining Flashcards
Nuclear staining done with basic dyes
Cationic dye that forms salt linkages with DNA or RNA
Nuclear staining done with dyes and mordants
Dye is combined with, or followed by, a metal mordant, which helps link the stain to the tissue
What is Isoelectric point (IEP) with respect to pH
IEP is the point at which positive and negative charges are equal, this is independent of pH
This is important because most stains depend on ionic, covalent, or hydrogen bonds to bind the dye to the tissue
Thus the pH of a stain can be adjusted to moderate its effect
Proteins are cationic below IEP pH6, anionic above IEP pH6
so eosin (anionic -) is kept at pH5 so proteins remain cationic (+) when staining cytoplasm
Chromophore
Organic structures that confer color to a dye
Are easily reduced due to an affinity for Hydrogen(+)
Auxochrome
An ionizing group that enables the dye to link to tissue (can be on the dye or the mordant)
Mordant
A metal that helps bind the dye to a tissue
ex: aluminum, tungsten, iron, chromium, lithium
Lake
Dye + mordant = insoluble dye lake
Cationic Dye
Charge on the dye is positive
auxochrome is amino group NH2
Typically chloride salts
Anionic Dye
Charge on the dye is negative
auxochrome is sulfonic, carboxyl COOH, or hydroxyl OH
typically sodium salts
Amphoteric Dye
Cationic(+) if pH is below IEP, Anionic(-) if pH is above IEP
acidic environment causes the dye to be basic, basic environment causes the dye t be acidic
IEP varies by dye used, the iEP value is a pH
hematein
Acid Dyes
Charge on the dye is negative (-)
Auxochromes are sulfonic, carboxyl (-COOH), and Hydroxyl (-OH) groups
Eosin, acid fuchsin, picric acid, light green, Orange G
Basic Dyes
Charge on the dye is positive (+)
Auxochrome is the Amino group (-NH2)
Alcian blue, crystal violet, safranin
Progressive Staining
Dye up to the desired intensity, then stop the reaction
Regressive Staining
tissue is intentionally overstained, then decolorized with a differentiator to achieve desired stain intensity on the element of interest
Commonly used for mordant dyes
Feulgen Reaction Purpose
Demonstrate DNA
Feulgen Reaction Principle
HCl hydrolyses DNA removing the purine (A and G, pure as gold) bases, while leaving behind sugars and phosphates
The resulting aldehyde (-CHO) can be demonstrated with Schiff reagent
Feulgen Reaction Fixative
Avoid strong acid fixatives like picric acid (Bouin’s) because they will overhydrolyze the DNA
Any fixative without a strong acid is appropriate
Feulgen Reaction Basic Procedure
- Deparaffinize (xylene) and hydrate
- Rinse in 1N HCl (displace remaining xylene/water)
- Heat in 1N HCl for 8-12 minutes (Hydrolysis)
- Rinse in cold 1N HCl then in distilled water (stops hydrolysis)
- Stain in Schiff reagent for 1 hour (dye binds to aldehydes)
- Wash briefly
- Rinse in 3 changes off sulfurous acid (reducing agent deactivates any residual Schiff)
- Wash in running tap water for 5 minutes
- Counterstain with 1% aqueous light green
- Dehydrate, clear (xylene), coverslip
Feulgen Reaction Results
DNA (nuclei) is reddish purple
Counterstained cytoplasm is light green
Feulgen Reaction Technical Notes
The Feulgen reaction must be carefully monitored because it has 2 phases, if the reaction runs too long you will get a false negative result
- removal of purine bases and formation of aldehyde (-CHO) groups
- removal of histones and apurinic acids
Duration of Feulgen reaction is dependent on fixative used
Counterstain must be applied lightly to avoid masking the Feulgen reaction
Methyl-Green Pyronin-Y Purpose
Differentiate between DNA and RNA
Identify plasma cells or immunoblasts in tissues (smears, cores, aspirates)
MGP Principle
DNA and RNA have different degrees of polymerization which results in differential staining
DNA stains with Methyl Green
RNA stains with Pyronin (Rose)
Methyl Green outcompetes pyronin for DNA binding sites
MGP Preferred Fixative
10% Neutral buffered formalin
B-5, B+, Zenker, and Helly are also acceptable
Carnoy is the best
MGP Basic Procedure
- Deparaffinize, hydrate
- Stain with methyl green pyronin-y solution for 5 minutes
- Rinse with distilled water
- Blot dry with filter paper
- Dip in 2 changes of acetone
- Dip in 1:1 acetone:xylene
- Dip in 2 changes Xylene
- Incubate in Xylene 5 minutes
- Coverslip
MGP Results
DNA: green to blue-green Nuclei: Green to blue-green Goblet cells: mint green RNA: red to rose Background: pale pink or colorless Immunoblast and plasma cell cytoplasm: Intense RED
Polychromatic stain
A compound dye or dye mixture that contains components of different colors
typically used for bone marrow specimens
Romanowsky Stain
Polychrome stain category commonly used in pathology of blood and bone marrow samples
May-Grunwald/Giemsa Stain Purpose
Differentiate cells in blood generating tissues, esp white blood cells, and demonstrate some microorganisms
Giemsa Principle
“Neutral” dyes combine basic(+) methylene blue with acidic(-) eosin resulting in a wide range of colors for staining blood smears
Often used to demonstrate white blood cells
Giemsa Preferred Fixative
Zenker or B-5 (mercuric)
10% NBF is acceptable
Giemsa Basic Procedure
- Deparaffinize and hydrate
- 5-10 minutes in Lugol Iodine to remove mercury from B-5/Zenker fixative, wash in tap water, treat with sodium thiosulfate, wash in running water, wash in distilled water
- 3 minutes 100% Methanol 2 times
- Stain in working Jenner solution for 5-6 minutes
- Transfer to working Giemsa for 45 minutes
- Rinse quickly
- Differentiate in 1% acetic water
- Rinse in distilled water
- Dehydrate, clear, coverslip
Giemsa Results
Nuclei: blue
Bacteria: blue
Leukocyte cytoplasm: pink, gray, blue depending on cell type and development
Giemsa Technical Notes
Working solutions are not stable, make fresh and discard for each use
pH of stain solutions are critical, optimize for the fixative being used
optimal pH of 6.4-6.9
Difference and preference between Synthetic and Natural Mounting Resins
Naturals are acidic, turn yellow, cause stain fading over time, and dry very slowly
Synthetics have the ideal refractive index, are neutral, dry quickly and don’t yellow with age
What is Aqueous mounting media and when is it used?
Used when dehydrating and clearing will adversely effect the stain (like Oil red O for lipids)
Has a lower refractive index so tissue doesn’t appear as transparent
Refractive index and relation to transparency
Refractive index measures the bending of light as it passes from one medium to another
You want the refractive index of your mounting media as close as possible to that of your tissue to maximize transparency and clarity
Refractive index of tissue
1.53-1.54
Refractive index of mounting medias
Synthetic Resin: 1.51-1.55
Aqueous: 1.41-1.43
MGP Technical Notes
Ethyl green is better because you don’t have to purify it with chloroform
Carnoy fixed tissue yields the best results
Acids/some fixatives can depolymerize DNA preventing green stain
Pyronin isn’t specific to RNA, rather it is out competed for DNA by methyl green
Chloroform hazardous waste disposal
5 Factors that affects dye binding
- pH affects charges on dyes and target molecules
- increased temperature tends to increase rate of staining
- Higher concentration of dye molecules tends to increase dye binding
- salts other than the dye can increase or decrease stain intensity of certain tissue components
- Fixative components can effect the ability of dyes to bind
