Multiplex immunofluorescence staining of coverslip-mounted paraffin-embedded tissue sections

Area of Science:

• Biomedical Research
• Histology
• Immunofluorescence Microscopy

Background:

• Animal and human tissues are crucial for physiological and pathophysiological research.
• Ethical concerns and limited availability necessitate maximizing tissue utilization.
• Existing methods often limit the amount of information obtainable from a single tissue section.

Purpose of the Study:

• To develop a new method for multiplex immunofluorescence (IF) staining enabling multiple uses of the same kidney tissue section.
• To optimize a protocol for sequential staining and destaining of paraffin-embedded kidney tissues.
• To enhance the efficiency and information retrieval from precious tissue samples.

Main Methods:

• Paraffin-embedded kidney sections were mounted on coated coverslips.
• Multiplex IF staining was performed over five iterative rounds, each including antibody labeling, imaging, and antibody removal via stripping buffer.
• Final round involved hematoxylin and eosin staining for tissue morphology.
• Imaging was conducted using a widefield epifluorescence microscope with a 60x oil objective.

Main Results:

• The method successfully labeled tubular segments, blood vessels, and interstitial cells in kidney tissue.
• Confocal-like resolution was achieved with a conventional widefield epifluorescence microscope.
• Increased Z-resolution was obtained for paraffin-embedded tissues.
• Quantitative and spatial expression information for multiple proteins was retrieved from a single section.

Conclusions:

• This multiplex IF protocol offers a time-saving approach for analyzing multiple proteins and tissue morphology.
• The method maximizes the utility of valuable tissue samples, reducing the need for serial sections.
• It provides a simple, effective, and potentially supplementary protocol for standard IF staining in research settings.