Related Experiment Video
Updated: Jan 3, 2026
A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections
Published on: March 30, 2020
Simultaneous Polychromatic Immunofluorescent Staining of Tissue Sections and Consecutive Imaging of up to Seven
Alfonso J Schmidt1, Johannes U Mayer1, Paul K Wallace2
1Malaghan Institute of Medical Research, Wellington, New Zealand.
Insights
This study introduces a 7-color immunofluorescent staining protocol for confocal microscopy, enabling simultaneous detection of multiple targets. The enhanced method utilizes novel tandem dyes and provides clear guidelines for optimizing antibody concentrations and correcting non-specific signals in multicolor imaging.
Area of Science:
- Life Sciences
- Microscopy
- Immunofluorescence
Background:
- Confocal microscopy is a vital tool in life sciences, with traditional methods relying on indirect immunofluorescence.
- Directly conjugated antibodies have increased the feasibility of multicolor staining, but limitations exist in the number of detectable parameters.
Purpose of the Study:
- To present an improved polychromatic immunofluorescent staining protocol for simultaneous detection of seven fluorescent parameters.
- To enable advanced multicolor imaging using standard confocal laser scanning microscopes.
Main Methods:
- Utilized a standard confocal laser scanning microscope with four laser lines and four photomultiplier tubes.
- Incorporated new tandem dyes (Brilliant Blue, Brilliant Violet) emitting in blue and violet regions.
- Developed a clear methodology for optimizing antibody concentrations and identifying/correcting non-specific signals.
Main Results:
- Successfully achieved simultaneous detection of seven fluorescent parameters.
- Demonstrated differentiation of multiple fluorochromes through the use of novel tandem dyes.
- Established guidelines for antibody titration and spillover optimization in complex multicolor staining.
Conclusions:
- The described protocol significantly enhances the multiplexing capability of standard confocal microscopy.
- This advancement allows for more comprehensive analysis of biological samples through increased fluorescent parameter detection.
- The protocol provides a robust framework for complex immunofluorescent imaging, aiding researchers in life sciences.
Abstract:
Confocal microscopy has been an important imaging tool for life scientists for over 20 years. Early techniques focused on indirect staining processes that involved staining with an unconjugated primary antibody, followed by incubation with a secondary fluorescent antibody that would reveal and amplify the signal of the primary antibody. With more and more directly conjugated fluorescent primary antibodies becoming commercially available, staining with multiple fluorescent primary antibodies is now more frequent. To date, staining with up to three primary antibodies and a nuclear dye is widely practiced. Here, we describe an important improvement to the standard polychromatic immunofluorescent staining protocol that allows the simultaneous detection of seven fluorescent parameters using a standard confocal laser scanning microscope with four laser lines and four photomultiplier tubes. By incorporating recently available tandem dyes that emit in the blue and violet regions of the visible light spectrum (Brilliant Blue and Brilliant Violet), we were able to differentiate several additional fluorochromes simultaneously. Due to the added complexity of 7-color immunofluorescent imaging, we developed a clear methodology to optimize antibody concentrations and simple guidelines on how to identify and correct non-specific signals. These are detailed in the following protocol. © 2019 by John Wiley & Sons, Inc. Basic Protocol: 7-Color immunofluorescent staining protocol using directly conjugated antibodies Support Protocol 1: Antibody titration protocol Support Protocol 2: Spillover optimization protocol.
