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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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

  1. Embed fresh tissue in OCT (optimal cutting temperature) solution and snap freeze using either dry ice or liquid nitrogen.
  2. Store tissues at -80 °C.

2. Cryosectioning

  1. 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

NameCompanyCatalog NumberComments
Acetone (histological grade)Fisher ScientificA16F-1GALFixing tissues
Alexa Fluor 488 anti-mouse CD3BioLegend100212Clone - 17A2; primary conjugated antibody
Alexa Fluor 488, eBioscience anti-human CD20ThermoFisher Scientific53-0202-82Clone - L26; primary conjugated antibody
Alexa Fluor 555 Mouse anti-Ki-67BD Biosciences558617Primary conjugated antibody
Alexa Fluor 594 anti-human CD3BioLegend300446Clone - UCHT1; primary conjugated antibody
Alexa Fluor 594 anti-mouse CD8aBioLegend100758Clone - 53-6.7; primary conjugated antibody
Alexa Fluor 647 anti-human CD8aBioLegend372906Clone - C8/144B; primary conjugated antibody
Alexa Fluor 647 anti-mouse CD206 (MMR)BioLegend141711Clone - C068C2; primary conjugated antibody
Alexa Fluor 647 anti-mouse CD4 AntibodyBioLegend100426Clone - GK1.5; primary conjugated antibody
C57BL/6 MouseCharles River Laboratories27Mouse frozen tissues used for multispectral training
Coplin JarSigma AldrichS6016-6EARehydrating and washing slides
DAPI SolutionBD Biosciences564907Nucleic Acid stain
Diamond White Glass Charged SlidesDOT ScientificDW7590WAdhering tissue sections
Dulbecco's Phosphate Buffered Saline 1x (without Ca and Mg)Fisher ScientificMT21031CVWashing and diluent
Gold Seal Cover SlipsThermoFisher Scientific3306Protecting stained tissues
Human Normal Tonsil OCT frozen tissue blockAMSBioAMS6023Human frozen tissue used for multispectral staining
Human Serum 1XGemini Bio-Products100-512Blocking and diluent for human tissues
inFormAkoya BiosciencesVersion 2.4.1Machine learning software
PerCP/Cyanine5.5 anti-human CD4BioLegend300529Clone - RPA-T4; primary conjugated antibody
PerCP-Cy 5.5 Rat Anti-CD11bBD Biosciences550993Clone - M1/70; primary conjugated antibody
PhenochartAkoya BiosciencesVersion 1.0.8Whole slide scan software
ProLong Diamond Antifade MountantThermoFisher ScientificP36965Mounting medium
Research CryostatLeica BiosystemsCM3050 SSectioning tissues
Superblock 1XThermoFisher Scientific37515Blocking mouse tissues
Tissue-Tek O.C.T SolutionSakura Finetek4583Embedding tissues
Vectra 3.0 Automated Quantitative Pathology Imaging System, 6 SlideAkoya BiosciencesCLS142568Semi-automated multispectral imaging system
Vectra SoftwareAkoya BiosciencesVersion 3.0.5Software to operate microscope

References

  1. 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).
  2. Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C.

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