Chapter 6: Nuclear and Cytoplasmic Staining

Question 1

Nuclear staining done with basic dyes

Answer 1

Cationic dye that forms salt linkages with DNA or RNA

Question 2

Nuclear staining done with dyes and mordants

Answer 2

Dye is combined with, or followed by, a metal mordant, which helps link the stain to the tissue

Question 3

What is Isoelectric point (IEP) with respect to pH

Answer 3

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

Question 4

Chromophore

Answer 4

Organic structures that confer color to a dye

Are easily reduced due to an affinity for Hydrogen(+)

Question 5

Auxochrome

Answer 5

An ionizing group that enables the dye to link to tissue (can be on the dye or the mordant)

Question 6

Mordant

Answer 6

A metal that helps bind the dye to a tissue

ex: aluminum, tungsten, iron, chromium, lithium

Question 7

Lake

Answer 7

Dye + mordant = insoluble dye lake

Question 8

Cationic Dye

Answer 8

Charge on the dye is positive
auxochrome is amino group NH2
Typically chloride salts

Question 9

Anionic Dye

Answer 9

Charge on the dye is negative
auxochrome is sulfonic, carboxyl COOH, or hydroxyl OH
typically sodium salts

Question 10

Amphoteric Dye

Answer 10

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

Question 11

Acid Dyes

Answer 11

Charge on the dye is negative (-)
Auxochromes are sulfonic, carboxyl (-COOH), and Hydroxyl (-OH) groups
Eosin, acid fuchsin, picric acid, light green, Orange G

Question 12

Basic Dyes

Answer 12

Charge on the dye is positive (+)
Auxochrome is the Amino group (-NH2)
Alcian blue, crystal violet, safranin

Question 13

Progressive Staining

Answer 13

Dye up to the desired intensity, then stop the reaction

Question 14

Regressive Staining

Answer 14

tissue is intentionally overstained, then decolorized with a differentiator to achieve desired stain intensity on the element of interest
Commonly used for mordant dyes

Question 15

Feulgen Reaction Purpose

Answer 15

Demonstrate DNA

Question 16

Feulgen Reaction Principle

Answer 16

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

Question 17

Feulgen Reaction Fixative

Answer 17

Avoid strong acid fixatives like picric acid (Bouin’s) because they will overhydrolyze the DNA
Any fixative without a strong acid is appropriate

Question 18

Feulgen Reaction Basic Procedure

Answer 18

1. Deparaffinize (xylene) and hydrate
2. Rinse in 1N HCl (displace remaining xylene/water)
3. Heat in 1N HCl for 8-12 minutes (Hydrolysis)
4. Rinse in cold 1N HCl then in distilled water (stops hydrolysis)
5. Stain in Schiff reagent for 1 hour (dye binds to aldehydes)
6. Wash briefly
7. Rinse in 3 changes off sulfurous acid (reducing agent deactivates any residual Schiff)
8. Wash in running tap water for 5 minutes
9. Counterstain with 1% aqueous light green
10. Dehydrate, clear (xylene), coverslip

Question 19

Feulgen Reaction Results

Answer 19

DNA (nuclei) is reddish purple

Counterstained cytoplasm is light green

Question 20

Feulgen Reaction Technical Notes

Answer 20

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

1. removal of purine bases and formation of aldehyde (-CHO) groups
2. 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

Question 21

Methyl-Green Pyronin-Y Purpose

Answer 21

Differentiate between DNA and RNA

Identify plasma cells or immunoblasts in tissues (smears, cores, aspirates)

Question 22

MGP Principle

Answer 22

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

Question 23

MGP Preferred Fixative

Answer 23

10% Neutral buffered formalin
B-5, B+, Zenker, and Helly are also acceptable
Carnoy is the best

Question 24

MGP Basic Procedure

Answer 24

1. Deparaffinize, hydrate
2. Stain with methyl green pyronin-y solution for 5 minutes
3. Rinse with distilled water
4. Blot dry with filter paper
5. Dip in 2 changes of acetone
6. Dip in 1:1 acetone:xylene
7. Dip in 2 changes Xylene
8. Incubate in Xylene 5 minutes
9. Coverslip

Question 25

MGP Results

Answer 25

``` 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 ```

Question 26

Polychromatic stain

Answer 26

A compound dye or dye mixture that contains components of different colors typically used for bone marrow specimens

Question 27

Romanowsky Stain

Answer 27

Polychrome stain category commonly used in pathology of blood and bone marrow samples

Question 28

May-Grunwald/Giemsa Stain Purpose

Answer 28

Differentiate cells in blood generating tissues, esp white blood cells, and demonstrate some microorganisms

Question 29

Giemsa Principle

Answer 29

"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

Question 30

Giemsa Preferred Fixative

Answer 30

Zenker or B-5 (mercuric) | 10% NBF is acceptable

Question 31

Giemsa Basic Procedure

Answer 31

1. Deparaffinize and hydrate 2. 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. 3 minutes 100% Methanol 2 times 4. Stain in working Jenner solution for 5-6 minutes 5. Transfer to working Giemsa for 45 minutes 6. Rinse quickly 7. Differentiate in 1% acetic water 8. Rinse in distilled water 9. Dehydrate, clear, coverslip

Question 32

Giemsa Results

Answer 32

Nuclei: blue Bacteria: blue Leukocyte cytoplasm: pink, gray, blue depending on cell type and development

Question 33

Giemsa Technical Notes

Answer 33

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

Question 34

Difference and preference between Synthetic and Natural Mounting Resins

Answer 34

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

Question 35

What is Aqueous mounting media and when is it used?

Answer 35

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

Question 36

Refractive index and relation to transparency

Answer 36

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

Question 37

Refractive index of tissue

Answer 37

1.53-1.54

Question 38

Refractive index of mounting medias

Answer 38

Synthetic Resin: 1.51-1.55 | Aqueous: 1.41-1.43

Question 39

MGP Technical Notes

Answer 39

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

Question 40

5 Factors that affects dye binding

Answer 40

1. pH affects charges on dyes and target molecules 2. increased temperature tends to increase rate of staining 3. Higher concentration of dye molecules tends to increase dye binding 4. salts other than the dye can increase or decrease stain intensity of certain tissue components 5. Fixative components can effect the ability of dyes to bind