Optimized Workflow for Iterative Bleaching Extends Multiplexity Imaging of Highly Autofluorescent Clinical Samples

Summary

The implementation of a low-cost, versatile photoirradiation technique with the manual IBEX method allows for optimal imaging of human tissue with significant native autofluorescence. This protocol details how to obtain multiplexed, whole-slide images from archived clinical samples using an inverted microscope, widely available reagents, and open-source software for image alignment and processing.

Abstract

The prevalence of pulmonary disease due to nontuberculous mycobacteria (NTM) has been increasing globally. Though NTM-induced pulmonary disease often presents with bronchiectasis, lung nodules, and cavitary disease, the host response associated with these distinct pulmonary injury patterns has not been well characterized in situ. We sought to evaluate mechanisms of NTM-induced pathology by performing deep phenotypic analysis of immune cell populations in lung tissue from individuals with NTM disease in comparison to a gold standard of granulomatous inflammation, tuberculous (TB) lung disease.

Formalin-fixed, paraffin-embedded (FFPE) lung blocks from patients with either disseminated NTM, pulmonary NTM, or pulmonary TB disease were sectioned and stained following a modified version of the Iterative Bleaching Extends multi-pleXity (IBEX) imaging method. Optimization of the IBEX method was first required to address the significant endogenous fluorescence of lung sections due to the high density of collagen, elastin, and erythrocytes native to pulmonary tissue that is worsened by FFPE preservation and the age of the FFPE blocks. In this protocol, we detail a photoirradiation protocol and modified antigen retrieval method that reduces the autofluorescence of clinically archived samples.

Furthermore, we provide guidance on antibody panel design, including common challenges such as cross-reactivity, epitope loss following photoirradiation, spectral overlap between adjacent fluorophores, and low signal-to-noise for certain targets. To facilitate efficient imaging of whole lung tissue sections, slides were imaged with a widefield microscope and postprocessed with open-source software to improve image quality. Together, this workflow addresses a critical need by documenting how to obtain highly multiplexed, high-resolution, whole-slide images from challenging samples using widely available instrumentation and reagents. Beyond quantitative imaging of clinical samples, these techniques can be applied to diverse sample types to overcome both endogenous and exogenous sources of fluorescence.

Introduction

Tuberculosis (TB) is one of the leading causes of death, with approximately 1.3 million people dying from TB infection each year¹. Though not as lethal as tuberculosis, there has been a significant increase in pulmonary infections and subsequent disease caused by nontuberculous mycobacteria (NTM) in recent years². At the tissue level, the host immune response to pulmonary Mycobacterium tuberculosis (Mtb) infection has been well studied and includes the formation of granulomas, heterogeneous aggregates of lymphocytes, dendritic cells, macrophages, and neutrophils³. While granulomatous infl....

Protocol

Archived autopsy and surgical resection lung tissue sections were collected under protocol 09-H-0172 (NCT00943514), Natural History of Bronchiectasis, at the National Institutes of Health Clinical Center Laboratory of Pathology. This protocol was IRB-approved, and the participants provided informed consent.

1. Automated antigen retrieval

NOTE: Deparaffinization and antigen retrieval were performed using an automated research staining platform (Figure 1). Previous work determined that a dual antigen retrieval approach consisting of 30 min with a citrate-based pH 6 epitope retri....

Results

Designing a multiplexed antibody-based imaging panel requires significant time, effort, and resources¹². These challenges are especially pronounced when designing a panel for archived FFPE samples from highly autofluorescent tissues as described here (Figure 1). To overcome these challenges, we first identified the brightest fluorophores compatible with our dye inactivation protocol (Table 1). For example, Alexa Fluor Plus secondary antibodies are rep.......

Discussion

Clinical lung pathology from pulmonary tuberculosis is characterized by necrotizing granulomatous inflammation. NTM pulmonary pathology primarily occurs in two distinct patient populations with two separate pathologic patterns: isolated lung injury marked by granulomatous inflammation with bronchiolar obliteration and cavity formation in immunocompetent individuals, or diffuse nodular disease affecting the lungs and other organs in immunodeficient individuals^13,14

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.3% Triton-X-100	Sigma-Aldrich	T8787	Solution for blocking buffer
1% BSA	Sigma-Aldrich	A4503	Solution for blocking buffer
1% Human BD Fc Block	BD Biosciences	564220	Fc Block required for blocking buffer
150 W LED lamp	Amazon	B07VH3CVSF	LED light source for tissue photoirradiation
1x PBS	Gibco	10010-023	Solution for blocking buffer and wash steps
20 Gallon Storage Container	Amazon	B001B1C4G0	Container for photoirradiation process
40 W RGBW flood LED lamp	Amazon	B08QFPJSMD	RGB light source for tissue photoirradiation
Ag85B	Abcam	ab43019/NA	Antibody for IBEX imaging. See Table 1.
Alpha-smooth muscle actin DyLight 755	Novus Biologicals	NBP2-345221R/NA	Antibody for IBEX imaging. See Table 1.
Avidin/Biotin Blocking Kit	Abcam	ab64212	Avidin and biotin blocking solution
BD Cytofix/Cytoperm	BD Biosciences	554714	Fixation solution
BOND Dewax Solution	Leica Biosystems	NC0221076	Deparaffinization solution
BOND Epitope Retrieval Solution 1	Leica Biosystems	NC0235529	Citrate based pH 6 epitope retrieval solution (ER1)
BOND Epitope Retrieval Solution 2	Leica Biosystems	NC0235530	EDTA based pH 9 epitope retrieval solution (ER2)
BOND Research Detection System	Leica Biosystems	NC0396648	Automated research staining reagent
BOND RX Research Stainer	Leica Biosystems	NA	Automated research staining platform
BOND Wash Solution	Leica Biosystems	NC0221077	Wash buffer
CD15	BD Biosciences	347420/AB_400298	Antibody for IBEX imaging. See Table 1.
CD20 eFluor 660	Thermo Fisher Scientific	50-0202-82/AB_11150959	Antibody for IBEX imaging. See Table 1.
CD4 Alexa Fluor 488	R&D Systems	FAB8165G/AB_2728839	Antibody for IBEX imaging. See Table 1.
CD45 Phycoerythrin (PE)	Novus Biologicals	NBP2-34528PE/NA	Antibody for IBEX imaging. See Table 1.
CD68 iFluor 594	Caprico Biotechnologies	1064135/AB_2892745	Antibody for IBEX imaging. See Table 1.
Donkey anti-goat IgG Alexa Fluor Plus 488	Thermo Fisher Scientific	A32814/AB_2762838	Antibody for IBEX imaging. See Table 1.
Donkey anti-mouse IgG Alexa Fluor Plus AF647	Thermo Fisher Scientific	A32787/ AB_2762830	Antibody for IBEX imaging. See Table 1.
Donkey anti-mouse IgM Alexa Fluor 488	Jackson ImmunoResearch	715-545-020/AB_2340844	Antibody for IBEX imaging. See Table 1.
Donkey anti-rabbit IgG Alexa Fluor Plus AF555	Thermo Fisher Scientific	A32794/AB_2762834	Antibody for IBEX imaging. See Table 1.
EasyDip Staining System	Newcomer Supply	5300KIT	Slide staining system
Fluoromount-G	Thermo Fisher Scientific	00-4958-02	Mounting media
Hoechst	Biotium	40046	Nuclear stain for IBEX imaging. See Table 1.
Imaris File Converter x64 10.2.0	Imaris Oxford Instruments		Free file converter
ImarisViewer software x64 10.2.0	Imaris Oxford Instruments		Free image processing software
ImmEdge Pen	Vector Laboratories	H-4000	Hydrophobic barrier pen
Lithium Borohydride (1 gram)	STREM Chemicals	93-0397	Chemical for dye inactivation, buy small aliquots of this chemical (1 gram)
Lumican Biotin	R&D Systems	BAF2846	Antibody for IBEX imaging. See Table 1.
Micro Cover Glasses	VWR	48393-241	Glass slide covers
Pan-cytokeratin Alexa Fluor 750	Novus Biologicals	NBP2-33200AF750/AB_2868569	Antibody for IBEX imaging. See Table 1.
PELCO BioWave Pro Microwave System	Ted Pella Inc	36500-230	Non-heating scientific microwave
PELCO SteadyTemp Pro	Ted Pella Inc	50062	Fully automated temperature control for scientific microwave
SimpleITK	Open source software		Registration and channel processing software
Streptavidin Alexa Fluor 594	Thermo Fisher Scientific	S11227	Labeling reagent for IBEX imaging. See Table 1.
Widefield Microscope	Leica Microsystems	NA	Leica Microsystems THUNDER imager with LED8 light source and LAS X software (3.7.1.21655). For fluorescence imaging, a custom quad-band filter with external filter wheel (PN: 11536075) with two additional single-band filters (PN: 8118215) were used to image seven dye channels per pass. The filter excitation, dichroic and emission lines are (1) Quad-Band cube: dichroic at 391/32, 479/33, 554/24, 638/31, no excitation or emission filters; (2) external filter wheel position 1: 434/32, position 2: 520/40, position 3: 585/20, position 4: 720/60, position 5: pass-through; (3) single-band 1—585/22 excitation, 594 dichroic, 625/30 emission; (4) single-band 2—635/20 excitation, 647 dichroic, 667/30 emission.
Zeiss Optical Lens Cleaner	Amazon	B00GPVQVCO	Microscope objective lens cleaner
Antibodies
Ag85B	Abcam	ab43019	Polyclonal Rabbit IgG, cycle 1
Alpha-smooth muscle actin DyLight 755	Novus Biologicals	NBP2-345221R	1A4/asm-1, cycle 1
CD4 Alexa Fluor 488	R&D Systems	FAB8165G	Polyclonal Goat IgG, cycle 1
CD8 Alexa Fluor 647	BioLegend	372906	C8/144B, cycle 1
CD68 iFluor 594	Caprico Biotechnologies	1064135	KP1, cycle 1
Donkey anti-goat IgG Alexa Fluor Plus 488	Thermo Fisher Scientific	A32814	Polyclonal Donkey, cycle 1
Donkey anti-mouse IgG Alexa Fluor Plus AF647	Thermo Fisher Scientific	A32787	Polyclonal Donkey, cycle 1
Donkey anti-rabbit IgG Alexa Fluor Plus AF555	Thermo Fisher Scientific	A32794	Polyclonal Donkey, cycle 1
Hoechst	Biotium	40046	Nuclear label, cycle 1
CD15	BD Biosciences	347420	MMA, cycle 2
CD20 eFluor 660	Thermo Fisher Scientific	50-0202-82	L26, cycle 2
CD45 Phycoerythrin (PE)	Novus Biologicals	NBP2-34528PE	2B11 + PD7/26, cycle 2
Donkey anti-mouse IgM Alexa Fluor 488	Jackson ImmunoResearch	715-545-020	Polyclonal Donkey, cycle 2
Lumican Biotin	R&D Systems	BAF2846	Polyclonal Goat IgG, cycle 2
Pan-cytokeratin Alexa Fluor 750	Novus Biologicals	NBP2-33200AF750	AE-1/AE-3, cycle 2
Streptavidin Alexa Fluor 594	Thermo Fisher Scientific	S11227	N/A, cycle 2
Hoechst	Biotium	40046	cycle 2
Solutions
Blocking Buffer (Cycle 1)
Triton-X-100	Sigma-Aldrich	T8787	0.3% by volume
BSA	Sigma-Aldrich	A4503	1% by volume
Human BD Fc Block	BD Biosciences	564220	1% by volume
1x PBS	Gibco	10010-023	97.7% by volume
Primary Antibody Staining Solution #1 (Cycle 1)
Blocking Buffer (Cycle 1)	See above		Top up to volume after adding antibodies
Hoechst	Biotium	40046	1:5000
CD8 AF647	BioLegend	372906	1:10
Primary Antibody Staining Solution #2 (Cycle 1)
Blocking Buffer (Cycle 1)	See above		Top up to volume after adding antibodies
CD4 AF488	R&D Systems	FAB8165G	1:10
Ag85B	Abcam	ab43019/NA	1:100
Secondary Antibody Staining Solution #1 (Cycle 1)
Blocking Buffer (Cycle 1)	See above		Top up to volume after adding antibodies
Donkey Anti-Goat AF Plus 488	Thermo Fisher Scientific	A32814/AB_2762838	1:100
Donkey anti-Rabbit AF Plus 555	Thermo Fisher Scientific	A32794/AB_2762834	1:100
Donkey anti-Mouse AF Plus 647	Thermo Fisher Scientific	A32787/AB_2762830	1:50
Primary Antibody Staining Solution #3 (Cycle 1)
Blocking Buffer (Cycle 1)	See above		Top up to volume after adding antibodies
CD68 iF594	Caprico Biotechnologies	1064135	1:10
a-SMA DL755	Novus Biologicals	NBP2-345221R/NA	1:30
BSA-free Blocking Buffer (Cycle 2)
Triton-X-100	Sigma-Aldrich	T8787	0.3% by volume
Human BD Fc Block	BD Biosciences	564220	1% by volume
1x PBS	Gibco	10010-023	98.7% by volume
Primary Antibody Staining Solution #4 (Cycle 2)
BSA-free Blocking Buffer (Cycle 2)	See above		Top up to volume after adding antibodies
CD15	BD Biosciences	347420	1:30
Lumican Biotin	R&D Systems	BAF2846	1:100
CD45 PE	Novus Biologicals	NBP2-34528PE	1:20
CD20 eFluor 660	Thermo Fisher Scientific	50-0202-82	1:10
Pan-Cytokeratin AF750	Novus Biologicals	NBP2-33200AF750	1:10
Secondary Antibody Staining Solution #2 (Cycle 2)
BSA-free Blocking Buffer (Cycle 2)	See above		Top up to volume after adding antibodies
Donkey anti-Mouse IgM AF488	Jackson ImmunoResearch	715-545-020/AB_2340844	1:100
Strepavidin AF594	Thermo Fisher Scientific	S11227	1:200