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Reversing the effect of formalin on the binding of propidium iodide to DNA
1Department of Pediatrics, Cooper Hospital/UMC, Camden, New Jersey 08103.
Abstract:
Formalin, an excellent preservative of cellular morphology, is a commonly used fixative for tissue specimens in hospital pathology laboratories. This preserved material is a potential source of tissue for diagnostic and retrospective research studies on DNA using flow cytometry. Unfortunately, formalin interferes with the binding of propidium iodide (PI) and other fluorescent dyes to DNA, thus altering the measurement of DNA content by flow cytometry or image analysis. This interference has been attributed to the cross-linking of histones by formalin. Since formalin alters the measurement of DNA content in formalin-fixed and formalin-fixed, paraffin-embedded tissues, this study was designed to explore the use of various physicochemical methods to reverse the effect of the formalin on the binding of PI to DNA. This study demonstrates that resuspending formalin-fixed cells in PBS and heating them at 75 degrees C for at least 1 h prior to staining with PI restores the staining of the DNA to approximately the same fluorescence intensity as that of fresh tissue.
Insights
Formalin fixation hinders DNA measurement in pathology. Heating formalin-fixed cells in PBS at 75°C for 1 hour restores propidium iodide (PI) DNA staining for accurate flow cytometry analysis.
Area of Science:
- Biomedical research
- Molecular pathology
- Cellular analysis
Background:
- Formalin is a standard fixative for preserving tissue morphology in pathology.
- Formalin-fixed tissues are valuable for DNA analysis using flow cytometry.
- Formalin interferes with DNA staining by propidium iodide (PI), affecting measurement accuracy.
Purpose of the Study:
- To investigate methods to reverse formalin's interference with DNA-dye binding.
- To optimize DNA content measurement in formalin-fixed tissues for flow cytometry.
Main Methods:
- Formalin-fixed cells were treated using various physicochemical methods.
- Cells were resuspended in Phosphate-Buffered Saline (PBS).
- Heating at 75°C for at least 1 hour was applied prior to PI staining.
Main Results:
- A heat treatment protocol was developed to reverse formalin's effect on DNA.
- Restored PI staining intensity was comparable to fresh tissue.
- This method enhances DNA measurement accuracy in formalin-fixed samples.
Conclusions:
- Physicochemical treatment, specifically heat, can overcome formalin-induced DNA staining interference.
- This technique improves the reliability of DNA content analysis in archival pathology specimens.
- Enables more accurate retrospective DNA research using formalin-fixed tissues.