1. SPECIAL STAINS
FOR NUCLEIC ACID

2. Histochemistry: Based on chemical reactions between cell components and stains.
The end products of the reaction are permanent, colored precipitates that can be viewed under the microscope.
There are stains specific to each component of the cell, based on the basic or acidic nature of the dye.

3. Basic Principles of Histochemistry
Histochemistry combines the methods of histology with those of chemistry or biochemistry, to reveal the biochemical composition of tissues and cells beyond the acid-base distribution shown by standard staining methods (H & E), without disrupting the normal distribution of the chemicals.

4. Application Identify, quantify, and localize Chemical substances
Gene expression
Biological structures, organelles
Specific cell types
Clarify cell and tissue structure and morphology.

5. Limitations Of the Current Methods
Cannot be used for real time in vivo analysis of any tissue (requires the removal and killing of the tissue).
Uses in humans limited to biopsied tissues.
Tissue preparation and histo-chemical analysis may alter specimen morphology or chemistry depending on the methods and materials used.

6. The goal of Histochemistry
Presentation of Normal Chemical Distribution.
Presentation of Normal Chemical Composition.
Specificity of the Reaction.
Detectability of the Reaction Product.
Insolubility of the Reaction Product

7. Nucleic Acids
The nucleic acids, DNA and RNA, can be localized by specific and non specific methods.
DNA is found mainly in nuclei, and its amount is much the same in every cell.
RNA is found both in nuclei and in cytoplasm, and its amount varies widely, depending on a cell's functional state.

10. Cont.
DNA STAINING TECHNIQUES need to be improved to achieve more accurate data.
Conventional histological procedures can preserve tissue for morphological characterization, but not necessarily for macromolecules.
Analysis of DNA plays an important role in molecular biology, pathology, histochemistry, and immunohistochemistry.
A cell can be quantified by measuring total DNA content, normal or abnormal, in its nucleus

11. Objectives Explain the methods used in demonstrating nucleic acids.
Describe the techniques and principles of the methods used.

12. Staining used for N.A
Feulgen's reaction: determine the amounts of DNA.
Methyl Green Pyronin Stain to determine DNA and RNA.
Acridine orange: The fluorescence is yellow green if the complex contains DNA and red-orange if it contains RNA.

13. Feulgen Stain
The Feulgen nuclear reaction for the specific staining of DNA in cytohistochemical samples in situ was introduced by Feulgen and Rossenbeck in 1924.
These authors devised a chemical treatment, HCI hydrolysis, to produce free aldehyde groups in the DNA backbone structure that could be detected by a colored reaction for aldehydes.

14. Figure 1: Photomicrograph showing Feulgen stained nuclei of normal oral mucosa (Feulgen stain, x200)

17. FEULGEN STAIN SOLUTIONS USED ARE :
1M HCL acid used for acid hydrolysis to break the purine-deoxyribose bond and yield an aldehyde.
- Done at 60̊ C (HCL should be preheated to 60 ̊ C )
- Time (minutes) depends upon the fixative used
- For carnoy’s and formalin – 8 minutes used
B) Schiff reagent - The aldehydes are then demonstrated by schiff’s reagent
C) Bisulfite solution
RESULT DNA : red-purple
CYTOPLASM : green

18. DNA BY FEULGEN STAIN

19. METHYL GREEN PYRONIN Y
Methyl Green-Pyronin Stain is for identification of Plasma Cells in Tissue Sections. Methyl Green-Pyronin Stain is for “In Vitro Diagnostic use.”
Methyl Green-Pyronin (MGP) is used to demonstrate plasma cells and RNA in tissue sections and cytologic preparations. The procedure is a simplified method for use with formalin fixed tissue or alcohol-ether fixed smears.
Pyronin stains the cytoplasm of plasma cells and most nucleoli red.

20. METHYL GREEN PYRONIN METHOD
Reagents :
Methyl green
- impure dye contains methyl violet – removed by washing with chloroform
- pure methyl green specific for DNA
- NH2 of dye reacts with phosphate of DNA
Pyronin
- binds to any negatively charged tissue constituent
- apart from RNA, binds to acid mucins and cartilage
RESULTS – DNA & cytoplasm: green-blue
RNA : red

23. Depapffinization by Xylene Hydration by Alcohol
Staining by MGP for 5-30 min.
Dehydration by n-butyl alcohol for 5min x2.
Clear by toluene for 5min.