Nucleic acid stains

stains for nucleic acids

1. NUCLEIC ACID STAINS

2. NUCLEIC ACIDS
●Two types:
–DNA – inside the nucleus
–RNA – in the cytoplasm
●Composition
–Sugar ( Deoxyribose/ Ribose)
–Phosphate
–Nitrogenous base

3. ●Demonstration of nucleic acids depends on
–Reaction of dyes to phosphate group
–Aldehydes derived from the sugar group
(deoxyribose)
●Histochemical methods can not detect
nitrogenous bases.

4. ●Methods of demonstration of Nucleic acids
–Fulgen technique ( demonstrates sugar in DNA)
–Methyl green pyronin technique ( demonstrates
phosphates)
–Acridine orange (by fluorescent method)
–Gallocyanin-chrome alum method

5. ●Gallocyanin-chrome alum method do not
separate DNA and RNA ( stains both blue), so
suitable extraction method must be used.

6. ●Extraction can be done by
–Digestion method: deoxyribonuclease and
riboxynuclease can digest DNA and RNA.
–Chemical method:
●Perchloric acid – To remove RNA – 10% perchloric acid
at 4 degree C overnight
●Trichloroacetic acid
●Hydrochloric acid

7. Fuelgen Stain
●Principle
–Fuelgen method selectively stains DNA.
–Treatment with Hcl (acid hydrolysis) removes purin bases
and exposes the aldehydes in sugar. The aldehyde group
reacts with Schiffs reagent to produce a colored reaction.
–RNA can not be hydrolysed by Hcl so the fuelgen
reaction is DNA specific
●Result
–DNA : red – purple
–Cytoplasm - green

8. ●Procedure
–Fixation of the tissues in carnoys fixative or 10 % formalin.
–Treat the fixed tissue in 1N Hcl (preheated to 60 deg C) in a
water bath for 8 – 10 minutes
–Immediately transfer to Schiffs reagent at room temperature
for 30 min or the tissue stains deep purple.
–Dehydrate with ethanol
–Clear with xylene
–Mount using resinous medium.
●BISULFITE SOLUTION – NOT USED ANYMORE

10. Methyl Green Pyronin Method
●Principle
–Methyl green (obtained from methyl violet by
washing impurities with chloroform) is specific for
DNA. NH2 of the dye reacts with the phosphate of
the DNA to give a color reaction.
–Pyronin binds to the negatively charged component
of the tissue. Apart from RNA it also binds to acid
mucin and cartilage.

11. ●Results:
–DNA : green blue
–RNA : Red

12. ●Procedure
–Deparaffinize tissue section.
–Rinse in tap water
–Rinse thoroughly in distilled water
–Stain with methyl green pyronin stain for 2 – 7 min at room
temperature.
–Dip slides 1 -2 times each in 2 changes of distilled water
–Dehydrate – alcohol
–Clear -xylene
–Mount DPX

14. ●Uses
– For qualitative and quantitative estimation of DNA
and RNA
–For staining plasma cells in tissue sections – stains
cytoplasm red.

15. Ninhydrin – Schiff method
●Method of histochemical demonstration of protein
bound amino group by light microscopy.
●Principle
–Protein bound amino acids undergo oxidative deamination
after treatment with ninhydrin at neutral pH and 37 deg C
resulting in the formation of protein bound aldehyde groups.
–The aldehyde group reacts with Schiffs reagent to give a
visible color reaction.
●Result
–Amino acids : purplish pink

16. ●Fixation
–Neutral buffered formalin
–Formaldehyde vapor for freeze dried tissues
●Sections
–Paraffin
–Cryostat
–Freeze dried
●Solutions
–0.5% ninhydrin in absolute alcohol
–Schiffs reagent

17. ●Procedure
–Treat sections with 70 percent alcohol
–Treat with ninhydrin solution overnight at 37 deg C
–Wash in running tap water
–Schiffs reagent – 45 min
–Wash in running tap water
–Counterstain – alum hematoxylin
–Wash in tap water, dehydrate with alcohol, clear,
mount.

18. Millon reaction for Tyrosine
●Principle
–Compounds containing hydroxyphenyl group react
with Millons reagent to give red color reaction.
Tyrosine is the only amino acid containing
hydroxyphenyl group and Millons reaction is
specific for tyrosin.
–Tyrosine with reacted with acidified mercuric sulfate
produces yellow precipitate of mercury amino acid
complex. On addition of sodium nitrate in presence
of heat mercury amino acid complex changes to
mercury phenolate which is red in color.

19. ●Result
–Tyrosine containing protein : red or pink
●Solutions
–Solution a
●10g mercuric sulfate+ mixture of (10ml conc H2SO4 and
90 ml distilled water) → dissolved by heating.
●Cool at room temp
●Add 100 ml of distilled water.

20. –Solution b
●250 g of sodium nitrate dissolved in 10 ml of Distilled
water.
–Staining solution
●Prepared by adding 5 ml of solution b to 50 ml of solution
a.

21. ●Procedure
–Wash sections in water
–Immerse sections in staining solution in a beaker
and gently bring to boil. Simmer for two min.
–Cool to room temperature
–Wash in 3 changes of distilled water – 2 mins each.
–Dehydrate clear and mount.