5. Histochemistry. Methods of demonstration of Nucleic Acids?

Question 1

What affinity do NA have?

Answer 1

basophilic - affinity for bases

Question 2

MOD for DNA? Process? Staining results?

Answer 2

• Feulgen Reactions:
• Used to identify chromosomal material
• Process:
• Bring sections to water.
• Rinse sections in M - HCL at room temperature 1 min.
• Place sections in preheated M - HCL at 60°c (this hydrolysis time will vary and depends on the fixative. For formalin fixed tissues 8 mins hydrolysis is required.
• Rinse sections in M - HCL at room temperature 1 min.
• Transfer sections to Schiff’s reagent 45 min -1 hr.
• Rinse sections in bisulphite solution x 3 washes - 2 mins each.
• Rinse well in water.
• Counter stain with 1% light green in 1% acetic acid 40 sec.
• Dehydrate, clear. Mount sections in DPX

Staining Results:
-DNA - red

Question 3

MOD of RNA? Process? Staining results?

Answer 3

• Methyl green pyronin Y
• Process:
• Deparaffinise and hydrate to distilled water.
• Place on a staining rack. Flood sections with methyl green – pyronin Y solution. Let stand for 5 minutes.
• Rinse quickly in distilled water.
• Blot completely dry with blotting paper.
• Place in acetone for 10 seconds.
• Dip several times in equal parts acetone and xylene.
• Clear in xylene, three or four changes.
• Mount with synthetic resin.

Staining Results:

• DNA - blue to blue-green
• RNA - pink to red
• Background - pale pink

DNA and RNA can also be analysed by staining cell/tissue sections w/ basic stain.