Staining 4.2 : Nuclear And Cytoplasmic Staining

Question 1

Nucleus

Answer 1

1. Appearance based on cell cycle phase
   - interphase : membrane bound
   - mitosis: mitosis bodies
2. Contains:
   - nuclear membrane
   - nuclear pores
   - nucleolus
   - chromatin (euchromatin (doesn’t pick up stain), heterochromatin (stainable))
3. Stains with:
   - basic dyes
   - Anionic dyes combined with metal mordants (hematoxylin, carmine)

-

Question 2

Nuclear stains

Answer 2

• Natural stains:
  
  • carmine, brazilin, hematoxylin (CBH)

-Synthetic:
- chromogens
- Red: safranine O, Nuclear Fast Red, Neutral Red
- color variation due to mordant: Celestine blue, gallocyanine, gallamine blue, tango blue
- thiazines: this in, toluidine blue O, methylene blue
- violets: crystal violet , gentian violet
- greens: methyl green, ethyl green

• fluorochromes:
  
  • quinacrine, acridine orange, ethidium bromide, proprium bromide, DAPI, TOTO, YOYO

Question 3

Hematoxylin

Answer 3

• hemaoxylin dye powder must be oxidized to created hematein
• Al’s called ripening/aging/maturing

Two OH groups changed to =O
- loses 2 H+ in the process
- more -vely charged molecule

Question 4

Hematoxylin oxidation

Answer 4

1. Natural:
   - create solution, expose to light and air, store in clear glass bottle close to window, have bottle half full and uncap
   - takes 3-6 months to ripen
   - good for 2-3 years but will continue to ripen slowly
   - transfer to dark bottle to slow ripening; store in tightly sealed cabinet

E.g.
- Ehrlich, Delafield, PTAH

2. Chemical

• hastens ripening, usually immediately ready for use
• has short half life (continues to oxidize from air and light hematein (brown) converted to oxyhematein (colorless))
• store in dark bottle that is tightly capped, at room temp in dark cabinet

1. Sodium iodate (NaIO3)
   - Mayer, Gill
   2.Mercuric oxide (HgO2)
   - Harris (now has sodium iodate due to mercury toxicity)
2. Potassium Permanganate (KMnO4)
   - Mallory’s PTAH
3. Alcoholic Iodine
   - Cole
4. Hydrogen peroxide
   - Thomas
5. Potassium iodate
   - Carazzi

Question 5

Mordants

Answer 5

• hematein alone will not attach to nuclei
• more -vely charged, trying to attach to a negatively charged phosphate backbone of DNA
• use metal salt to attach hematein to DNA
• metal salt (mordant) + dye = lake

Mordant used will influence :
- components stained
- final color of hematoxylin

A. aluminum = Purple to blue
B. Iron = Blue-black
C. Chromium = Blue-black
D. Copper. = Blue-green to purple
E. Nickel = Violet shades
F. Tin = Red
G. Lead = Dark brown
H. Osmium = Green-brown

Question 6

Aluminum Hematoxylin

Answer 6

• IEP of nucleic acids is 1.5-2.0 (basophilic)
• pH of Al hematoxylin is 2.2 -2.9 so it is basic
  
  • acidify using:
    
    • acetic, citric acids and adding protons H+ to solution -> solution becomes reddish (lose purple blue appearance)

Question 7

Filtering hematoxylin

Answer 7

• some have to be filtered before use, eg Harris. Gill and Mayer do not need filtration
• if you see a green sheen on surface of hematoxylin:
  
  • overoxidized hematoxylin, need to be filtered away (oxy-hematein)
  • if don’t remove, will end up with precipitate on section and slide

Question 8

Al hematoxylin staining time

Answer 8

• acidic solution
  
  • free H+ in solution binds non specific Anionic components
• decreased background staining
• excess Al
  — helps counteract overoxidation of hematein by the chemical oxidizer in solution
  — must watch amount carefully
• too much aluminum can precipitate out on the slides

Question 9

progressive vs regressive

Answer 9

• most aluminum hematoxylins can be used either way
• progressive: leave in hematoxylin until nuclei are blue enough
• regressive:
  
  • need to differentiate
  • use weak acid (other side of IEP) (HCl, Hac)
  • can be made up in aqueous solution, or alcoholic solution
    — aqueous sol, less control - must do fast dips
    — alcoholic, more control - can leave in longer for longer period of time
  • better for automated stainers

Question 10

Bluing aluminum hematoxylin

Answer 10

• color short from red hematein to blue hematein
• creates blue-purple color
• in acidic solution (ph 2.5 hematoxylin)
  
  • H+ protons bind to metal-hematein bonds
• changes absorption of light
• appears red
• in higher PH
  
  • H+ is removed
  • color will shift to blue
  • will stay that way once removed from bluing agent
• cannot over blue slides
• pH dependent process, place in weak alkali solution
• ammonia water
• lithium carbonate
• Scott’s tap water
  
  • K bicarbonate 2g
  • Mg sulfate 2g
  • Deionised water 1L
• Tap H2O - increased temp; impurities

Question 11

Iron hematoxylin

Answer 11

• mordant also works as an oxidizer
• very easy to overoxidize with iron
  
  • mix together just before use
  • ready immediately
  • GOOD FOR FEW HOURS to FEW DAYS
• chemicals used:
  
  • +2 ferrous (chloride, sulfate)
  • +3 ferric (chloride, aluminum sulfate (ferric alum))
• used as nuclear stain when
  
  • succeeding stains are lengthy
  • succeeding stains are very acidic (iron is more resistant to leaving tissue); revomoes aluminum hematoxylin
  • purple/blue will not contrast with background

Question 12

Iron hematox

Answer 12

• by varying reagents, time, temperature, pH and differentiator, can get any of the following to stain:
• nuclei
• muscle striations
• cell organelles
• Protozoa
• myelin sheaths
• elastin