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A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections
In This Article
Summary
We describe a rapid staining method to perform multispectral imaging on frozen tissues.
Abstract
Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.
Introduction
Recent advances in microscopic imaging techniques have significantly improved our knowledge and understanding of biological processes and disease states. In situ detection of proteins in tissues via chromogenic immunohistochemistry (IHC) is routinely performed in pathology. However, detection of multiple markers using chromogenic IHC staining is challenging1 and newer methods to use multiplex immunofluorescence (mIF) staining approaches, wherein multiple biological markers are labeled on a single tissue sample, are being developed. The detection of multiple biological markers is useful, because information related to tissue architecture, spatia....
Protocol
Mouse spleen and HLF16 mouse tumor tissues23 were obtained from our laboratory. Human tonsil tissue was purchased from a commercial vendor. Details are provided in the Table of Materials.
1. Tissue Embedding
- Embed fresh tissue in OCT (optimal cutting temperature) solution and snap freeze using either dry ice or liquid nitrogen.
- Store tissues at -80 °C.
2. Cryosectioning
- Cut 8 _.......
Representative Results
Detection of single-stained markers on frozen spleen sections
As the semiautomated imaging system uses a liquid crystal tunable filter (LCTF) system that allows for a wider range of wavelength detection25, and because no signal amplification steps were performed here, we first optimized the detection of our primary-conjugated antibodies for each marker on the microscope. An example is shown in Figure 1, where each single-stained marker is pseudo.......
Discussion
Frozen tissues have extensively been used for mIF imaging to traditionally detect three to four markers31 on a tissue using the direct and indirect method32. In the direct method, antibodies are conjugated to fluorescing dyes or quantum dots33 to label the tissue, whereas in the indirect method, an unconjugated primary antibody is used to label the tissue followed by a fluorophore-conjugated secondary antibody that specifically recognizes the primary.......
Acknowledgements
Imaging and analysis guidance was provided by the Research Resources Center – Research Histology and Tissue Imaging Core at the University of Illinois at Chicago established with the support from the office of the Vice Chancellor for Research. The work was supported by NIH/NCI RO1CA191317 to CLP, by NIH/NIAMS (SBDRC grant 1P30AR075049-01) to Dr. A. Paller, and by support of the Robert H. Lurie Comprehensive Cancer Center to the Immunotherapy Assessment Core at Northwestern University.
....Materials
| Name | Company | Catalog Number | Comments |
| Acetone (histological grade) | Fisher Scientific | A16F-1GAL | Fixing tissues |
| Alexa Fluor 488 anti-mouse CD3 | BioLegend | 100212 | Clone - 17A2; primary conjugated antibody |
| Alexa Fluor 488, eBioscience anti-human CD20 | ThermoFisher Scientific | 53-0202-82 | Clone - L26; primary conjugated antibody |
| Alexa Fluor 555 Mouse anti-Ki-67 | BD Biosciences | 558617 | Primary conjugated antibody |
| Alexa Fluor 594 anti-human CD3 | BioLegend | 300446 | Clone - UCHT1; primary conjugated antibody |
| Alexa Fluor 594 anti-mouse CD8a | BioLegend | 100758 | Clone - 53-6.7; primary conjugated antibody |
| Alexa Fluor 647 anti-human CD8a | BioLegend | 372906 | Clone - C8/144B; primary conjugated antibody |
| Alexa Fluor 647 anti-mouse CD206 (MMR) | BioLegend | 141711 | Clone - C068C2; primary conjugated antibody |
| Alexa Fluor 647 anti-mouse CD4 Antibody | BioLegend | 100426 | Clone - GK1.5; primary conjugated antibody |
| C57BL/6 Mouse | Charles River Laboratories | 27 | Mouse frozen tissues used for multispectral training |
| Coplin Jar | Sigma Aldrich | S6016-6EA | Rehydrating and washing slides |
| DAPI Solution | BD Biosciences | 564907 | Nucleic Acid stain |
| Diamond White Glass Charged Slides | DOT Scientific | DW7590W | Adhering tissue sections |
| Dulbecco's Phosphate Buffered Saline 1x (without Ca and Mg) | Fisher Scientific | MT21031CV | Washing and diluent |
| Gold Seal Cover Slips | ThermoFisher Scientific | 3306 | Protecting stained tissues |
| Human Normal Tonsil OCT frozen tissue block | AMSBio | AMS6023 | Human frozen tissue used for multispectral staining |
| Human Serum 1X | Gemini Bio-Products | 100-512 | Blocking and diluent for human tissues |
| inForm | Akoya Biosciences | Version 2.4.1 | Machine learning software |
| PerCP/Cyanine5.5 anti-human CD4 | BioLegend | 300529 | Clone - RPA-T4; primary conjugated antibody |
| PerCP-Cy 5.5 Rat Anti-CD11b | BD Biosciences | 550993 | Clone - M1/70; primary conjugated antibody |
| Phenochart | Akoya Biosciences | Version 1.0.8 | Whole slide scan software |
| ProLong Diamond Antifade Mountant | ThermoFisher Scientific | P36965 | Mounting medium |
| Research Cryostat | Leica Biosystems | CM3050 S | Sectioning tissues |
| Superblock 1X | ThermoFisher Scientific | 37515 | Blocking mouse tissues |
| Tissue-Tek O.C.T Solution | Sakura Finetek | 4583 | Embedding tissues |
| Vectra 3.0 Automated Quantitative Pathology Imaging System, 6 Slide | Akoya Biosciences | CLS142568 | Semi-automated multispectral imaging system |
| Vectra Software | Akoya Biosciences | Version 3.0.5 | Software to operate microscope |
References
- van der Loos, C. M. Chromogens in Multiple Immunohistochemical Staining Used for Visual Assessment and Spectral Imaging: The Colorful Future. Journal of Histotechnology. 33 (1), 31-40 (2010).
- Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C.
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