Some practical considerations in quantitative absorbance microspectrophotometry. Preparation techniques in DNA...

Area of Science:

• Histology
• Biochemistry
• Cell Biology

Background:

• Quantitative cytophotometry of DNA in tissue sections is crucial for various biological studies.
• Accurate DNA measurement relies heavily on consistent tissue preparation and staining protocols.

Purpose of the Study:

• To experimentally review Feulgen and gallocyanine-chrome-alum stains for DNA quantitative cytophotometry.
• To provide guidelines for optimal tissue preparation and staining for microspectrophotometry.

Main Methods:

• Evaluation of different fixatives (formalin, glutaraldehyde, ethanol, Bouin's, Carnoy's, methanol-formalin-glacial acetic acid) for Feulgen and gallocyanine-chrome-alum staining.
• Assessment of dye purity for Feulgen staining using absorbance peaks.
• Optimization of Feulgen staining pH for reproducibility.
• Determination of optimal tissue section thickness (4-8 microns) based on nuclear characteristics.
• Selection of appropriate mounting medium refractive index (1.54-1.56 for animal tissues).

Main Results:

• Formalin-fixed tissues are suitable for both stains; glutaraldehyde or ethanol fixation require modifications.
• Feulgen stain purity is confirmed by absorbance peaks (543-546 nm); pH 1.5 ensures reproducibility.
• Gallocyanine is preferred for Bouin's or glutaraldehyde-fixed tissues.
• Optimal section thickness is tissue-dependent, balancing nuclear overlap and full thickness.
• Mounting media refractive index matching tissue background is essential for accurate absorbance readings.

Conclusions:

• Specific fixation and staining protocols are critical for reliable DNA quantitative cytophotometry.
• Feulgen and gallocyanine-chrome-alum stains are effective for DNA measurement when optimized.
• Careful consideration of section thickness and mounting medium enhances quantitative accuracy.