A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

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 μm sections in a cryostat with temperatures set at -25 °C.
   NOTE: The preferred section thickness can be adjusted to gene....

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.......

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