Histochemical applications of two phenanthridinium compounds

Area of Science:

• Histochemistry
• Molecular Biology
• Biochemistry

Background:

• Nucleic acid staining is crucial for histochemistry.
• Ethidium compounds are fluorescent dyes used for nucleic acid detection.
• Differentiating DNA and RNA in fixed tissues presents challenges.

Purpose of the Study:

• To evaluate ethidium monoazide and ethidium bromide for nucleic acid staining in fixed tissues.
• To develop methods for selectively abolishing RNA or DNA fluorescence.
• To explore new histochemical approaches for nucleic acid analysis.

Main Methods:

• Staining of fixed, paraffin-embedded and fresh frozen rat and mouse tissues with ethidium compounds.
• Selective hydrolysis of RNA and DNA using varying acid concentrations and conditions.
• pH manipulation of staining solutions to differentiate nucleic acid fluorescence.

Main Results:

• Ethidium monoazide and ethidium bromide showed similar intense reactions with nucleic acids.
• RNA fluorescence was abolished by mild acid hydrolysis (1 N HCl, 5-min), while DNA required longer treatment.
• DNA fluorescence was preserved using anhydrous HCl in methanol, and selective RNA fluorescence abolition was achieved by lowering pH to 1.0.

Conclusions:

• Ethidium compounds are effective for nucleic acid staining in various tissue preparations.
• Selective acid hydrolysis and pH control provide methods to differentiate DNA and RNA fluorescence.
• This technique offers novel approaches for nucleic acid histochemistry and studying drug-DNA interactions.