Name: 반복적인 표백을 위해 최적화된 워크플로우는 고도의 자가형광 임상 샘플의 멀티플렉시티 이미징을 확장합니다. (동영상) | JoVE Journal
Uploaded: 2025-07-11T14:00:00.000+00:00
Duration: 6 min 52 s
Description: 학습하세요 광조사 기법을 활용한 임상 샘플의 자가형광 감소 및 면역세포 공간 분석 방법을. 이 프로토콜은 폐 조직의 결핵균 및 다양한 병리에서 면역세포 상호작용 연구에 활용됩니다.

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This work was supported by the Intramural Research Program of DIR/NIAID, NCI and NHLBI. Figure 1 was created using BioRender under an institutional license agreement with the NIH.

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<li>Quardokus, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organ+mapping+antibody+panels%3A+A+community+resource+for+standardized+multiplexed+tissue+imaging%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Organ mapping antibody panels: A community resource for standardized multiplexed tissue imaging.</a> Nat Methods. 20 (8), 1174-1178 (2023).</li>
<li>Mcshane, P. J., Glassroth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pulmonary+disease+due+to+nontuberculous+mycobacteria%3A+Current+state+and+new+insights%5BTitle%5D)+AND+%22Chest%22%5BJournal%5D)">Pulmonary disease due to nontuberculous mycobacteria: Current state and new insights.</a> Chest. 148 (6), 1517-1527 (2015).</li>
<li>Hsu, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutations+in+GATA2+are+associated+with+the+autosomal+dominant+and+sporadic+monocytopenia+and+mycobacterial+infection+%28monomac%29+syndrome%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Mutations in GATA2 are associated with the autosomal dominant and sporadic monocytopenia and mycobacterial infection (monomac) syndrome.</a> Blood. 118 (10), 2653-2655 (2011).</li>
<li>Vinh, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autosomal+dominant+and+sporadic+monocytopenia+with+susceptibility+to+mycobacteria%2C+fungi%2C+papillomaviruses%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Autosomal dominant and sporadic monocytopenia with susceptibility to mycobacteria, fungi, papillomaviruses.</a> Blood. 115 (8), 1519-1529 (2010).</li>
<li>Filipe-Santos, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((X-linked+susceptibility+to+mycobacteria+is+caused+by+mutations+in+nemo+impairing+CD40-dependent+IL-12+production%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">X-linked susceptibility to mycobacteria is caused by mutations in nemo impairing CD40-dependent IL-12 production.</a> J Exp Med. 203 (7), 1745-1759 (2006).</li>
<li>Galeotti, T., Van Rossum, G. D., Mayer, D. H., Chance, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+fluorescence+of+NAD%28P%29H+in+whole-cell+preparations+of+tumours+and+normal+tissues%5BTitle%5D)+AND+%22Eur+J+Biochem%22%5BJournal%5D)">On the fluorescence of NAD(P)H in whole-cell preparations of tumours and normal tissues.</a> Eur J Biochem. 17 (3), 485-496 (1970).</li>
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<li>Harman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lipofuscin+and+ceroid+formation%3A+The+cellular+recycling+system%5BTitle%5D)+AND+%22Adv+Exp+Med+Biol%22%5BJournal%5D)">Lipofuscin and ceroid formation: The cellular recycling system.</a> Adv Exp Med Biol. 266, 3-15 (1989).</li>
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A.J.R. is an employee at Leica Microsystems, Inc. The other authors declare no competing interests.

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 individuals13,14</sup...

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 inflammation is also a fundamental feature of host immunity to NTM infection, the immune cell populations and the spatial characteristics of this response remain understudied. Gaining a detailed understanding of the tissue components of immunopathology versus wound healing is critical for the development of treatments (e.g., host directed therapies) and preventative (i.e., vaccines) therapeutic strategies.
In this study, our team had access to rare, archived clinical samples acquired during lung resections or autopsies from patients with Mtb or NTM infection. The majority of these samples were over 10 years old and preserved as formalin-fixed, paraffin-embedded (FFPE) samples, restricting their use to histological assays. To overcome the marker limitations of traditional immunohistochemistry (IHC) and immunofluorescence (IF) imaging, we utilized the Iterative Bleaching Extends multi-pleXity&#160;(IBEX) method, an open-source, highly multiplexed imaging method4,5. Extension of the IBEX method to human lung FFPE samples required significant optimization due to high levels of endogenous fluorescence, a well described obstacle for multiplexed antibody-based imaging6.
Here, we provide a detailed protocol for reducing autofluorescence using a photoirradiation method optimized for lung FFPE tissue sections7,8. Importantly, the method described here utilizes an inexpensive, widely available light box that can be extended to other sample preparations with high autofluorescence. In addition to reducing autofluorescence prior to image acquisition, we provide guidance on antibody panel design such as pairing low abundance markers with bright fluorophore labels, evaluating the impact of photoirradiation on antibody labeling, and amplification with secondary antibodies in channels with high endogenous fluorescence. On average, we processed five slides corresponding to one tissue section, each ranging from 37 to 114 mm2. Finally, we demonstrate how to acquire whole-slide images using a widefield instrument. In summary, this protocol describes the adoption of IBEX to archived clinical samples and additionally offers guidance on how to troubleshoot issues commonly encountered with imaging highly autofluorescent samples.

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

optimized workflow for iterative bleaching extends multiplexity imaging of highly autofluorescent clinical samples

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

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.

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...

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.

The prevalence of pulmonary disease due to nontuberculous mycobacteria (NTM) has been increasing globally. Though NTM-induced pulmonary disease often ...

[Narrator] Using spatial proteomics, named Nature Methods' 2024 Method of the Year, the precise locations of immune cells along with cell-cell interactions can be mapped within tissues. This reveals spatial patterns linked to clinical outcomes. Building complex antibody panels and imaging tissues with high endogenous fluorescence present significant time, resources, and expertise challenges for IBEX and other fluorescence microscopy techniques. Using IBEX, tumor-specific features were identified in high-risk follicular lymphoma patients, and with Human Cell Atlas colleagues. a comprehensive spatial map of the human thymus was created. Little is known about the immune cell composition, spatial interactions, and cytokine production differences across diverse mycobacterial pulmonary pathologies. This protocol bridges this gap. This protocol offers a low-cost, adaptable photoirradiation method to significantly reduce broad-spectrum tissue autofluorescence, especially in difficult FFPE samples, while preserving antibody signal for spatial analysis. To begin, immerse the slides with tissue in PBS after completing antigen retrieval. Remove one slide from the PBS and use a lint-free wipe t

optimized-workflow-for-iterative-bleaching-extends-multiplexity

Research

JoVE Journal

Immunology and Infection

Background autofluorescence of biological samples often complicates fluorescence-based imaging techniques, especially in aged human postmitotic tissues. This protocol describes how autofluorescence from these samples can be effectively removed using a commercially available light emitting diode light source to photobleach the sample prior to immunostaining.

Simple Elimination of Background Fluorescence in Formalin-Fixed Human Brain Tissue for Immunofluorescence Microscopy

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Multiplex fluorescent immunohistochemistry is an emerging technology that enables the visualization of multiple cell types within intact formalin-fixed, paraffin embedded (FFPE) tissue. Presented are guidelines for ensuring a successful 7-color multiplex with instructions for optimizing antibodies and reagents, preparing slides, design and tips for avoiding common problems.

Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multiplex cyclic immunohistochemistry allows in situ detection of multiple markers simultaneously using repeated antigen-antibody incubation, image scanning, and image alignment and integration. Here, we present the operating protocol for identifying immune cell substrates with this technology in lung cancer and paired brain metastasis samples.

Multiplex Cyclic Fluorescent Immunohistochemistry

The combination of iterative-bleaching-extends-multiplexity (IBEX) and a commercial nucleotide labeling assay (Click-iT EdU) enables the detection and categorization of dividing cell types in highly dynamic processes in fixed frozen murine tissue sections. Furthermore, a novel open-source image processing pipeline provides high-throughput image acquisition and analysis.

High-throughput Imaging and Analysis Workflow for Evaluating Skin Cell Phenotypes and Proliferation States in Tissue Samples

Here, we describe the application of the Iterative Bleaching Extends Multiplicity (IBEX) method utilizing commercially available, automated tissue stainers and optimization considerations for the use of tyramide signal amplification-based reagents.

Iterative Bleaching Extends Multiplicity with Use of Staining Automation for Core Facilities

This article describes a protocol for multiplex immunofluorescence optimized to characterize the three-dimensional architecture of peritoneal metastases.

Three-Dimensional Imaging of Tumor-Bearing Tissue Using the Iterative Bleaching Extends Multiplexity Approach

Spectral Iterative Bleaching Extends Multiplexity (IBEX) builds upon the base IBEX technique by adding heparin blocking to minimize nonspecific binding and leveraging spectral detection with computational unmixing to suppress autofluorescence. This approach accelerates image acquisition while reducing sources of background, enabling robust multi-round, high-parameter spatial proteomic analyses.

High-plex Imaging using Spectral Confocal Microscopy to Minimize Non-specific Tissue Fluorescence

Multiplexed immunofluorescence enables sequential labeling of multiple antibody markers within a single tissue section to support spatial analysis of tissue architecture and cell populations. Here, a workflow for multiplex detection of senescence-associated, structural, and immune markers is presented, including manual and automated slide preparation, iterative imaging, and single-cell analysis using digital pathology software.

Multiplexed Immunofluorescence Assay for Spatial Assessment of Senescence Markers in vivo

학습하세요 광조사 기법을 활용한 임상 샘플의 자가형광 감소 및 면역세포 공간 분석 방법을. 이 프로토콜은 폐 조직의 결핵균 및 다양한 병리에서 면역세포 상호작용 연구에 활용됩니다.

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

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