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A Unique Immunofluorescence Protocol to Detect Protein Expression in Vascular Tissues: Tacking a Long Standing
1Department of Research, Bhopal Memorial Hospital & Research Centre, BHOPAL-38, INDIA.
Insights
This study presents an optimized immunofluorescence method to reduce autofluorescence in tissue samples, enhancing signal clarity for accurate protein detection in archival and cryo sections. The novel trio-methodology improves signal intensity and retention for reliable diagnostic imaging.
Area of Science:
- Histopathology
- Immunofluorescence Microscopy
- Biomedical Imaging
Background:
- Autofluorescence in formalin-fixed paraffin-embedded tissues reduces signal-to-noise ratio in immunofluorescence.
- Sources of autofluorescence include collagen, elastin, red blood cells, and lipofuscin, particularly in vascular and aging tissues.
- Aldehyde-fixation can also induce artifacts affecting fluorescence analysis.
Purpose of the Study:
- To develop an optimized indirect-immunofluorescence method for archival formalin-fixed paraffin-embedded tissues and cryo sections.
- To reduce autofluorescence interference and improve signal-to-noise ratio in immunofluorescence analysis.
- To achieve optimal fluorescence signals and imaging for accurate protein detection.
Main Methods:
- An optimized indirect-immunofluorescence protocol using a specific three-reagent combination was developed.
- The method was tested on archival formalin-fixed paraffin-embedded tissues and cryo sections, including various cancer types and non-malignant brain tissue.
- Sodium-borohydride, crystal violet, and Sudan black B were applied sequentially to mask autofluorescence.
Main Results:
- The combined application of sodium-borohydride, crystal violet, and Sudan black B effectively masked autofluorescence and non-specific background in vascular tissues.
- This trio-methodology significantly enhanced fluorescence signal intensity for target proteins.
- Quantifiable observations with maximized fluorescence signal intensity and longer retention time were achieved.
Conclusions:
- The developed trio-methodology is highly efficacious for masking autofluorescence in immunofluorescence analysis of archival tissues.
- This technique maximizes fluorescence signal intensity and ensures longer signal retention, even after prolonged storage.
- The method is applicable to various human tissues and protein targets for improved diagnostic imaging.
Objective:
Autofluorescence induced interference is one of the major drawbacks in immunofluorescence analysis of formalin-fixed paraffin-embedded tissues, as it decreases the signal-to-noise ratio of specific labeling. Apart from aldehyde-fixation induced artifacts; collagen and elastin, red blood cells and endogenous fluorescent pigment lipofuscin are prime sources of autofluorescence in vascular and aging tissues. We describe herein, an optimized indirect-immunofluorescence method for archival formalin-fixed paraffin-embedded tissues tissues and cryo sections, using a combination of 3-reagents in a specific order, to achieve optimal fluorescence signals and imaging.
Material And Method:
Human telomerase reverse transcriptase, a protein implicated as a proliferation marker, was chosen relevant to its expression in solid tumors along with 3 other intracellular proteins exhibiting nuclear and/or cytoplasmic expression. Staining was performed on 10 glioma tissue sections along with 5 of their cryo sections, 5 sections each of hepatocellular, lung, papillary-thyroid and renal cell carcinoma, with 10 non-malignant brain tissue samples serving as control. Specimens were imaged using epifluorescence microscopy, followed by software-based quantification of fluorescence signals for statistical analysis and validation.
Results:
We observed that the combined application of sodium-borohydride followed by crystal violet before antigen retrieval and a Sudan black B treatment after secondary antibody application proved to be most efficacious for masking autofluorescence/non-specific background in vascular tissues.
Conclusion:
This unique trio-methodology provides quantifiable observations with maximized fluorescence signal intensity of the target protein for longer retention time of the signal even after prolonged storage. The results can be extrapolated to other human tissues for different protein targets.
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