简介：
Overview
This article presents a low-cost photoirradiation technique that enhances imaging of human tissue with native autofluorescence. The protocol allows for multiplexed, whole-slide imaging from archived clinical samples using widely available reagents and open-source software.
Key Study Components
Area of Science
- Neuroscience
- Immunology
- Fluorescence Microscopy
Background
- Spatial proteomics enables mapping of immune cells and their interactions within tissues.
- Challenges exist in building antibody panels and imaging tissues with high endogenous fluorescence.
- IBEX technique identifies tumor-specific features in high-risk lymphoma patients.
- Little is known about immune cell composition in mycobacterial pulmonary pathologies.
Purpose of Study
- To provide a protocol that reduces tissue autofluorescence while preserving antibody signals.
- To facilitate spatial analysis of immune cells in complex tissue samples.
- To bridge knowledge gaps in immune cell interactions across various pathologies.
Methods Used
- Slides with tissue are immersed in PBS post-antigen retrieval.
- Blocking buffer and primary antibody solutions are prepared and applied.
- Photoirradiation is performed using specific LED lamps to reduce autofluorescence.
- Whole-slide imaging is conducted to visualize immune cell interactions and tissue features.
Main Results
- Significant reduction in autofluorescence was achieved after photoirradiation.
- Granulomas and immune cell types were visualized in lung tissue.
- Specific antibodies detected Mycobacterium tuberculosis near necrotic cores.
- IBEX imaging revealed detailed anatomical features of lung tissues.
Conclusions
- The protocol enhances the imaging of complex tissues while minimizing autofluorescence.
- It provides a valuable tool for studying immune cell interactions in various diseases.
- This method can be adapted for a wide range of clinical applications.
What is the main advantage of the photoirradiation technique?
It significantly reduces tissue autofluorescence while preserving antibody signals for better imaging.
How does this protocol aid in studying immune cells?
It allows for detailed mapping of immune cell interactions within tissues, providing insights into their spatial patterns.
What types of samples can be used with this protocol?
Archived clinical samples, particularly those with high endogenous fluorescence, can be effectively imaged.
What are the key components needed for the photoirradiation process?
A 150-watt LED lamp, a 40-watt RGBW flood LED lamp, and a large plastic container are essential.
How long does the photoirradiation process take?
The process involves an initial two-hour exposure followed by a 16-hour incubation period.
What types of immune cells were identified in the study?
CD15-positive neutrophils, CD20-positive B cells, CD4-positive T cells, and CD68-positive macrophages were visualized.
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