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Method Article

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

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DOI:

10.3791/67980

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July 11th, 2025

 ,  ,  ,  ,  ,  , 
1Critical Care Medicine and Pulmonary Branch, National Heart, Lung and Blood Institute, National Institutes of Health, 2Lymphocyte Biology Section and Center for Advanced Tissue Imaging, Laboratory of Immune System Biology, NIAID, NIH, 3Leica Microsystems Inc., 4T Lymphocyte Biology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health

In This Article

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 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 (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.

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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 retrieval solution (ER1) and then 30 min with an EDTA-based pH 9 epitope retrieval solution (ER2) yielded the best immunolabeling results for FFPE tissues9. Comparable results may be obtained using manual dewaxing and dual antigen retrieval using other described methods and support provided by the IBEX Imaging Community10,11.

  1. Gather slides and automated antigen retrieval supplies (slide covers, research detection system, citrate-based pH 6 epitope retrieval solution (ER1), EDTA-based pH 9 epitope retrieval solution (ER2), etc).
  2. Turn on the instrument, allow it to initialize for 20 min, place the research detection arm into the instrument, and insert reagent containers into the bottom of the machine.
  3. Open the software, click slide set up and select add study, and create a new name for the study.
  4. Select add slide and label the slides as desired.
  5. To follow this protocol, perform a Bake and Dewax protocol with a HIER protocol consisting of 30 min of ER1 and 30 min of ER2.
  6. Print labels and place them on top of the slides. Remove the black slide rack from the instrument and place the slides of interest on the rack. Place clear slide covers on each slide, return the slide rack to the instrument, and press the button below the rack to prompt the instrument to perform barcode scanning of the slides.
  7. Select play | ok and let the automated antigen retrieval commence with an approximate duration of 3 h.

2. Antibody labeling prior to photoirradiation

  1. After antigen retrieval is complete, immerse the slides in PBS.
    NOTE: Certain epitopes are sensitive to photoirradiation and become degraded after overnight exposure to the LED. Sensitive epitopes must be determined empirically and when discovered, immunolabeling of these epitopes should occur at this step. Ensure that the slide stays wet at all times following antigen retrieval.
  2. Remove one slide from PBS and dry off all excess PBS on the slide using a lint-free wipe; be careful not to touch the tissue. Draw a border around the tissue with a hydrophobic pen. Repeat the process for all remaining slides. Let the hydrophobic barrier dry for 10 min.
  3. Prepare a Slide Moisture Chamber by placing a small amount of water in the bottom of each well. Place slides in the Slide Moisture Chamber. Wick off PBS from the tissue without directly touching the tissue specimen.
  4. Add 200 µL of blocking buffer (Table of Materials) to each slide. Place the Slide Moisture Chamber in a non-heating scientific microwave with a mechanism for maintaining a steady temperature.
  5. Block non-specific antibody labeling by executing the following program established previously4: 2-1-2-1-2-1-2-1-2 program, where "2" denotes 2 min at 100 W and "1" denotes 1 min at 0 W. Execute the above program once for blocking. If this instrument is not available, block the tissue sections (5-10 µm) for 1 h at 37 °C as described previously5.
  6. Create Primary Antibody Staining Solution #1 (Cycle 1) (Table of Materials) during blocking buffer incubation. Prepare antibody mixes according to the Table of Materials. Gently mix the antibody cocktail and spin down the antibody mix with a mini centrifuge at 2,680 × g for 30 s.
    NOTE: This staining solution contains 1:5000 dilution of Hoechst that should be sufficient for the duration of the two-cycle experiment. Users may need to titrate Hoechst and other antibodies to obtain optimal results for their tissues and imaging configuration.
  7. Remove the Slide Moisture Chamber from the microwave when the cycle has completed and wick off the blocking buffer solution from each tissue. Add 200 µL of Primary Antibody Staining Solution #1 (Cycle 1) (Table of Materials) to each slide. Place the chamber with slides back into the microwave and run the primary antibody program for approximately 30 min: execute 2-1-2-1-2-1-2-1-2 program twice for primary antibody labeling.
    NOTE: Here, 200 μL of primary antibody solution is an estimate. Use an adequate volume of solution to cover the entire surface of the tissue section.
  8. Once the primary antibody labeling step is complete, wash each slide with 1,000 µL of PBS by tilting the slide vertically, pipetting PBS onto the tissue, and letting the PBS run off. Repeat this step 3-5x.
  9. Wick off excess PBS and fix the slides with 1% paraformaldehyde (PFA) for 10 min at room temperature in the humidity chamber.
    NOTE: PFA fixation can mask epitopes, preventing antibodies from binding their intended targets. When establishing an antibody panel for the first time, we recommend evaluating the impact of a gentle fixation (1% PFA for 10 min) by comparing the labeling pattern of tissue sections treated with or without fixative before immunolabeling. If an antibody is sensitive to 1% PFA fixation, then move this antibody before the postfixation step.
  10. Wash the slides extensively with 1,000 µL of PBS 3-5x. Leave PBS on the tissue until the photoirradiation step.

3. Tissue photoirradiation

  1. To prepare the photoirradiation box (Figure 2), obtain a 150 W LED lamp, a 40 W RGBW flood LED lamp, a large plastic container (20 gallon [75.71 L] volume or more), fresh 1x PBS, and a Petri dish.
  2. Place the Petri dish in the center of the 150 W lamp and fill it with 1x PBS. Use enough volume to immerse the slides in PBS. Perform the photoirradiation process in the cold room to minimize heat generation from lamps.
  3. Remove the slides from the Slide Moisture Chamber and place them in a Petri dish, ensuring that all slides are submerged in PBS.
  4. Place the 40 W lamp directly on top of the Petri dish with the light source facing the slides. Ensure that the 40 W lamp is set to red light setting prior to placement.
  5. Turn on both the 150 W and 40 W lamps. Cover the photoirradiation setup with the plastic container lid. Maintain the 40 W lamp on the red light setting for 2 h, then switch to the green light setting via the included remote for the remaining 16 h.
    NOTE: It is imperative to perform photoirradiation at approximately 4 °C to prevent unwanted heat distortion of the slide tissue. Pause point: photoirradiation can be performed overnight and the protocol can be resumed the following morning.

4. Antibody labeling post photoirradiation

  1. Remove the slides from the Petri dish and submerge them in 1x PBS in a Coplin jar, 50 mL conical tube, or a bucket from the slide staining system. Prepare the Primary Antibody Staining Solution #2 (Cycle 1) (Table of Materials) at recommended dilutions (Table 1). Use 200 µL of the solution for each slide.
  2. Wick off excess PBS from each individual slide with lint-free wipe making sure to dry the area around tissue without touching the tissue itself. Place the slides in the Slide Moisture Chamber.
  3. Gently vortex Primary Antibody Staining Solution #2 (Cycle 1) and add 200 µL to each slide, ensuring the entire tissue is covered.
  4. Place the Slide Moisture Chamber with the slides back into the microwave and run the primary antibody program for approximately 30 min as detailed in step 2.7
  5. During primary antibody labeling, make the Secondary Antibody Staining Solution #1 (Cycle 1) solution (Table of Materials).
  6. Once the microwave program has completed, remove the Slide Moisture Chamber and wash each slide 3-5x with 1,000 µL of 1x PBS.
  7. Wick off excess PBS from each individual slide with lint-free wipe, vortex the secondary antibody staining solution, and add 200 µL to each slide.
  8. Place the Slide Moisture Chamber in the microwave and run the secondary antibody program: A 2-1-2-1-2-1-2-1-2 program, where " 2" denotes 2 min at 100 W and " 1" denotes 1 min at 0 W. Execute the above program once for secondary antibody labeling.
  9. Wash the slides extensively 3-5x with 1 mL of 1x PBS and wick off excess PBS from each slide.
  10. Add Primary Antibody Staining Solution #3 (Cycle 1) containing directly conjugated antibodies to each slide. Perform antibody staining using the microwave or alternative (1 h at 37 °C) as outlined in step 2.7.
  11. Wash the slides again 3-5x with 1 mL of 1x PBS and carefully wick off excess PBS from each slide.
  12. Add 30 µL of mounting medium directly to each tissue section and place a coverslip covering the entirety of the tissue section. Inspect the slide for air bubbles and push air bubbles to the perimeter of the slide with gentle pressure. Repeat the process for the remaining slides.
  13. Remove excess mounting medium with a lint-free wipe and use optical lens cleaner to clean any residue remaining on the surface of the coverslip. Allow all slides to set for 10-15 min at room temperature prior to performing image acquisition on a microscope.

5. Microscopy setup and image acquisition

  1. Turn on the widefield microscope and LED8 light source; launch the software. Select the objective (here, a 20x/0.8 dry objective). Place the camera in 16-bit mode in the configuration tab. Select the required channels based on the fluorophores present in the antibody panels (see Table 1 for further guidance).
    NOTE: The following steps are specified for the referenced microscope. An equivalent widefield microscope with a comparable light source and filter cubes for excitation and emission of several fluorophores and a precision stage could be used.
  2. Clean the coverslip with lens cleaner and lens paper immediately before imaging. Visually inspect the coverslip and remove any dust or lint from the surface before imaging. Position the slide firmly into the top left corner (or equivalent) of the stage insert, so the tissue will be consistently positioned in the XY plane (critical for the alignment of cyclic images).
  3. Set up the acquisition settings by creating a channel corresponding to each fluorophore included in the antibody panel. Select the corresponding filter cube and LED for each fluorophore. Adjust the intensity of each LED and the exposure times for each channel. See Table 1 for a list of the image settings used here.
  4. Before image acquisition, select the Hoechst channel and turn on Linked Shading. Use the Linked Shading wizard to set this for the objective selected.
    NOTE: This protocol details how to perform whole slide images through the acquisition of large tile scans. Linked Shading or shading correction is needed to overcome uneven illumination across tiled images.
  5. In the software, click the Live button to visualize the Hoechst channel. Use the coarse focus knob to bring the tissue section into focus.
  6. Select each fluorescent channel and assess the tissue section for the expected staining pattern for all fluorescently labeled antibodies in the cycle.
  7. Select Navigator in the software. Using the Hoechst channel and spiral function in Navigator, map the entire tissue section or region of interest to be imaged. If multiple tissue sections will be imaged per slide, repeat this step to create a map of the tissue using Navigator.
  8. Identify the ROI(s) to be imaged using the polygon, magic wand, or other ROI selection tools in Navigator. Carefully draw the tissue outline to capture the fewest tiles needed to cover the desired ROI(s). Delete extra tiles to reduce the total acquisition time.
  9. Once ROIs are created, review and adjust channel settings to minimize saturated pixels and optimize signal-to-noise. Select the heatmap lookup table in the image display window. The colors indicate the fill level of pixels, with reds, oranges, yellows, and whites indicating easier-to-see variations in intensity; blue pixels indicate saturated pixels. To avoid losing information and compromising image quality, reduce the exposure time to minimize the number of saturated (blue) pixels.
  10. In Navigator, use the Focus Map tool to create a topographical map of the tissue. Set a Focus Map point every 3-4 FOVs for large tiles or uneven samples.
    NOTE: This step is critical for the large tile scans required for whole slide imaging. In general, the quality of the image scales with the number of Focus Map Points selected; however, this process can be quite time-consuming, especially for large tile scans.
    1. Create Focus Map Points in the order in which they will be scanned by the microscope. To match the tile-scanning order of the referenced microscope, select Focus Map Points along a serpentine path that matches the path used by the microscope.
    2. Set the first Focus Map Point using manual focus. Use autofocus for the remaining Focus Map Points. Sit and watch to ensure that the optimal focus is selected for each Focus Map Point. Manually adjust Focus Map Points as needed. Increase the range used to find each Focus Map Point for uneven and or large tissues.
  11. Set up the processing using the following parameters: Small Volume Computational Clearing (SVCC) with a Refractive Index (RI) set to match the mounting medium (here, 1.4); strength set to 98%, and feature size set to 2,000 nm for membrane markers or 3,000 nm for nuclear markers. Run the computational clearing protocol during image acquisition.
  12. Start the tile scan. Write down the tile number and keep the ROIs constant throughout all cycles.
  13. After image acquisition, merge the computationally cleared images with Mosaic Merge found in the Process tab, with the Smooth option selected. Visually inspect each image for stitching errors and repeat the Mosaic Merge with new parameters if tiling errors are present.
    NOTE: Poorly aligned images will prevent registration of iteratively acquired images.
  14. If needed, use the Dye Separation module to compensate for spectral spillover between channels as described previously5.

6. Cycles of dye inactivation, antibody labeling, and image acquisition

  1. After image acquisition has completed, immerse the slides in a 50 mL conical tube of sterile 1x PBS until the coverslips fall off. If the coverslip does not fall off, run the PBS directly over the slide or use tweezers to gently remove the coverslip.
  2. Prepare lithium borohydride (LiBH4) solution by measuring out 3-5 mg of powder into a plastic weigh boat. Add diH2O at a 1:1 ratio directly to the powder (e.g., 3.2 mg of LiBH4 and 3.2 mL of diH2O) and homogenize the solution with a pipette. Use a syringe to run the solution through a 0.2 µm filter into a 50 mL conical tube and record the concentration and exact time the solution was created. Allow the solution to incubate at room temperature for 3-5 min until large bubbles are seen throughout the solution.
    NOTE: We recommend using the dye inactivation solution within 4 h of preparation. CAUTION: Perform the following steps in a chemical fume hood and use appropriate personal protective equipment (PPE). This reaction can produce hydrogen gas, which is highly flammable. It is important to work with small amounts of LiBH4 (<10 mg) at all times. For further guidance, watch the tutorial videos on how to prepare LiBH4 safely on the IBEX Knowledge-Base10,11.
    Replace LiBH4 vial after 4 weeks of use due to decreased bleaching effectiveness after repeated exposure to air. We recommend buying small aliquots of LiBH4 (1 gram).
  3. Remove each slide from the 1x PBS soak, wick off excess PBS with lint-free wipe, and place it in the slide moisture chamber.
  4. Treat each non-coverslipped slide with LiBH4 for 30 min by pipetting the solution directly onto the slide with enough volume to cover the tissue. Remove old LiBH4 solution from each slide and refresh with a new aliquot of LiBH4 at 10 min and 20 min from the same LiBH4 stock solution.
  5. Wash the slides extensively 3-5x with 1x PBS. Use quick exchanges of PBS, soaking the slides for 1 min per wash.
  6. To acquire an image for background subtraction, place a new coverslip on each slide as described in steps 4.12-4.13 and acquire images of the tissue post-dye inactivation. Create a new project for the background subtraction image and repeat steps 5.2-5.14 to acquire the image. Image using the same number of tiles, channels, channel settings, and FOVs established for the cycle 1 image. Keep the Focus Map points in the same FOVs, but use autofocus to adjust each Focus Map point for this new image.
    NOTE: Use the channel that contains the repeated fiducial marker (Hoechst) to identify unique structures (nuclear shapes) that are present in the 'live' image (background subtraction image, for example) and the previous image (cycle 1).
  7. To minimize nonspecific fluorescent signals from biotinylated antibodies in the second antibody cycle, perform avidin and biotin blocking.
    1. Wick off excess PBS and apply avidin blocking solution to each slide at room temperature for 15 min.
    2. Wash the slides 3-5x with 1x PBS and wick off excess PBS when the washes are completed.
    3. Apply biotin blocking solution to each slide at room temperature for 15 min and wash the slides 3-5x with 1x PBS as in step 6.7.2.
  8. Prepare new BSA-free Blocking Buffer (Cycle 2) (Table of Materials) for 
    Primary Antibody Staining Solution #4 (Cycle 2) and Secondary Antibody Staining Solution #2 (Cycle 2).
    NOTE: These solutions will not require bovine serum albumin because BSA can compromise avidin/biotin blocking step.
  9. Dilute primary antibodies at manufacturer-recommended concentrations in the Primary Antibody Staining Solution #4 (Cycle 2). Gently vortex this solution and add to each slide within the humidity chamber, ensuring the tissue is completely covered with the primary antibody solution. Place the slide moisture chamber with the slides into the microwave and run the primary antibody program for approximately 30 min as detailed in step 2.7-2.8.
  10. Perform secondary antibody labeling with Secondary Antibody Staining Solution #2 (Cycle 2) as detailed in steps 4.6-4.9. Once staining has been completed, place a new coverslip on each slide as previously described in steps 4.12-4.13. Place the slide back onto the microscope stage and reposition the slide to be in the same XY plane for the previous images.
    NOTE: Use the channel that contains the repeated fiducial marker (Hoechst) to identify unique structures (nuclear shapes) that are present in the 'live' image (cycle 2, for example) and the previous image (cycle 1).
  11. Create a new project for cycle 2 image and repeat steps 5.2-5.14 to acquire the cycle 2 image. Image using the same number of tiles, channels, channel settings, and FOVs established for the cycle 1 image. Keep the Focus Map points in the same FOVs but use autofocus to adjust each Focus Map point for this new image. Once the cycle 2 image is acquired, proceed to image alignment or repeat steps in section 6 for additional cycles with directly conjugated antibodies. NOTE: Use the channel that contains the repeated fiducial marker (Hoechst) to identify unique structures (nuclear shapes) that are present in the 'live' image (cycle 2 image, for example) and the previous image (cycle 1). Additional cycles require directly conjugated antibodies because IBEX does not strip antibodies.

7. Image alignment

  1. Convert raw images from the microscope output .lif files into .ims files with the free File Converter and image processing with the open-source software as described previously5 and detailed in videos on the IBEX Knowledge-Base10,11.
  2. For each imaging cycle, edit the file and apply any desired processing steps (e.g., channel naming, channel pseudo-coloring).
    1. Perform background subtraction and channel thresholding using the channel arithmetics functions created for SimpleITK to decrease the autofluorescence signal (https://niaid.github.io/imaris_extensions/XTChannelArithmetic.html).
      NOTE: We perform these steps on each image (e.g., Cycle 1, Cycle 2) before aligning them into one composite image. This is because it is easier to work on the smaller, individual images than the larger combined image.
  3. Devise a consistent naming strategy to use the registration software. Name channels with a prefix-separator character-postfix (e.g. 'Cycle1 CD8_AF647') with a space as a separator character to denote the channel in which the anti-CD8 AF647 antibody was used in the first cycle.
    NOTE: See the help file extension for further details: https://niaid.github.io/imaris_extensions/XTRegisterSameChannel.html.
  4. After all channels have been appropriately named, launch the registration software, click Imaris Extensions | SimpleITK | Affine.
  5. Upload the .ims file for each cycle by using the browse button next to File names. Enter the Channel name prefix separator character. If following the example above, type a single space, then click Next.
  6. Select the registration channel used as the common fiducial marker between cycles (e.g. Hoechst).
  7. Select the Fixed Image, the image to which other images will be registered.
    NOTE: We recommend registering to Cycle 2 of iterative images.
  8. Select the desired directory for the 'Output File'. The Start registration at resolution option will be set at maximal resolution by default to allow the best registration possible. If computing power is limited, reduce this parameter.
    NOTE: For the data described here (12-15 GB per image) we used a maximal resolution of 5.
  9. Click Register to begin. Once registration has completed, wait for the Resample and Save Combined Image button to appear, click on it, and wait for the progress bar to go to completion.
  10. Open the completed output file in the processing software. The registered image with all cycles and channel information will be available and ready for analysis or publication.

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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...

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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 individuals13,14

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Disclosures

A.J.R. is an employee at Leica Microsystems, Inc. The other authors declare no competing interests.

Acknowledgements

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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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.3% Triton-X-100Sigma-AldrichT8787Solution for blocking buffer
1% BSASigma-AldrichA4503Solution for blocking buffer
1% Human BD Fc BlockBD Biosciences564220Fc Block required for blocking buffer
150 W LED lampAmazonB07VH3CVSFLED light source for tissue photoirradiation
1x PBS Gibco10010-023Solution for blocking buffer and wash steps
20 Gallon Storage ContainerAmazonB001B1C4G0Container for photoirradiation process
40 W RGBW flood LED lamp AmazonB08QFPJSMDRGB light source for tissue photoirradiation
Ag85BAbcamab43019/NAAntibody for IBEX imaging. See Table 1.
Alpha-smooth muscle actin DyLight 755Novus BiologicalsNBP2-345221R/NAAntibody for IBEX imaging. See Table 1.
Avidin/Biotin Blocking KitAbcamab64212Avidin and biotin blocking solution
BD Cytofix/CytopermBD Biosciences554714Fixation solution
BOND Dewax SolutionLeica BiosystemsNC0221076Deparaffinization solution
BOND Epitope Retrieval Solution 1Leica BiosystemsNC0235529Citrate based pH 6 epitope retrieval solution (ER1)
BOND Epitope Retrieval Solution 2Leica BiosystemsNC0235530EDTA based pH 9 epitope retrieval solution (ER2)
BOND Research Detection SystemLeica BiosystemsNC0396648Automated research staining reagent
BOND RX Research StainerLeica BiosystemsNAAutomated research staining platform
BOND Wash SolutionLeica BiosystemsNC0221077Wash buffer
CD15BD Biosciences347420/AB_400298Antibody for IBEX imaging. See Table 1.
CD20 eFluor 660Thermo Fisher Scientific50-0202-82/AB_11150959Antibody for IBEX imaging. See Table 1.
CD4 Alexa Fluor 488R&D SystemsFAB8165G/AB_2728839Antibody for IBEX imaging. See Table 1.
CD45 Phycoerythrin (PE)Novus BiologicalsNBP2-34528PE/NAAntibody for IBEX imaging. See Table 1.
CD68 iFluor 594Caprico Biotechnologies1064135/AB_2892745Antibody for IBEX imaging. See Table 1.
Donkey anti-goat IgG Alexa Fluor Plus 488Thermo Fisher ScientificA32814/AB_2762838Antibody for IBEX imaging. See Table 1.
Donkey anti-mouse IgG Alexa Fluor Plus AF647Thermo Fisher ScientificA32787/ AB_2762830Antibody for IBEX imaging. See Table 1.
Donkey anti-mouse IgM Alexa Fluor 488Jackson ImmunoResearch715-545-020/AB_2340844Antibody for IBEX imaging. See Table 1.
Donkey anti-rabbit IgG Alexa Fluor Plus AF555Thermo Fisher ScientificA32794/AB_2762834Antibody for IBEX imaging. See Table 1.
EasyDip Staining SystemNewcomer Supply5300KITSlide staining system
Fluoromount-GThermo Fisher Scientific00-4958-02Mounting media
HoechstBiotium40046Nuclear stain for IBEX imaging. See Table 1.
Imaris File Converter x64 10.2.0Imaris Oxford InstrumentsFree file converter
ImarisViewer software x64 10.2.0Imaris Oxford InstrumentsFree image processing software
ImmEdge PenVector LaboratoriesH-4000Hydrophobic barrier pen
Lithium Borohydride (1 gram)STREM Chemicals93-0397Chemical for dye inactivation, buy small aliquots of this chemical (1 gram)
Lumican BiotinR&D SystemsBAF2846Antibody for IBEX imaging. See Table 1.
Micro Cover GlassesVWR48393-241Glass slide covers
Pan-cytokeratin Alexa Fluor 750Novus BiologicalsNBP2-33200AF750/AB_2868569Antibody for IBEX imaging. See Table 1.
PELCO BioWave Pro Microwave SystemTed Pella Inc36500-230Non-heating scientific microwave
PELCO SteadyTemp ProTed Pella Inc50062Fully automated temperature control for scientific microwave
SimpleITKOpen source softwareRegistration and channel processing software
Streptavidin Alexa Fluor 594Thermo Fisher ScientificS11227Labeling reagent for IBEX imaging. See Table 1.
Widefield MicroscopeLeica MicrosystemsNALeica 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 CleanerAmazonB00GPVQVCOMicroscope objective lens cleaner
Antibodies
Ag85BAbcamab43019Polyclonal Rabbit IgG, cycle 1
Alpha-smooth muscle actin DyLight 755Novus BiologicalsNBP2-345221R1A4/asm-1, cycle 1
CD4 Alexa Fluor 488R&D SystemsFAB8165GPolyclonal Goat IgG, cycle 1
CD8 Alexa Fluor 647BioLegend372906C8/144B, cycle 1
CD68 iFluor 594Caprico Biotechnologies1064135KP1, cycle 1
Donkey anti-goat IgG Alexa Fluor Plus 488Thermo Fisher ScientificA32814Polyclonal Donkey, cycle 1
Donkey anti-mouse IgG Alexa Fluor Plus AF647Thermo Fisher ScientificA32787Polyclonal Donkey, cycle 1
Donkey anti-rabbit IgG Alexa Fluor Plus AF555Thermo Fisher ScientificA32794Polyclonal Donkey, cycle 1
HoechstBiotium40046Nuclear label, cycle 1
CD15BD Biosciences347420MMA, cycle 2
CD20 eFluor 660Thermo Fisher Scientific50-0202-82L26, cycle 2
CD45 Phycoerythrin (PE)Novus BiologicalsNBP2-34528PE2B11 + PD7/26, cycle 2
Donkey anti-mouse IgM Alexa Fluor 488Jackson ImmunoResearch715-545-020Polyclonal Donkey, cycle 2
Lumican BiotinR&D SystemsBAF2846Polyclonal Goat IgG, cycle 2
Pan-cytokeratin Alexa Fluor 750Novus BiologicalsNBP2-33200AF750AE-1/AE-3, cycle 2
Streptavidin Alexa Fluor 594Thermo Fisher ScientificS11227N/A, cycle 2
HoechstBiotium40046cycle 2
Solutions
Blocking Buffer (Cycle 1)
Triton-X-100Sigma-AldrichT87870.3% by volume
BSASigma-AldrichA45031% by volume
Human BD Fc BlockBD Biosciences5642201% by volume
1x PBS Gibco10010-02397.7% by volume
Primary Antibody Staining Solution #1 (Cycle 1)
Blocking Buffer (Cycle 1)See aboveTop up to volume after adding antibodies
HoechstBiotium400461:5000
CD8 AF647BioLegend3729061:10
Primary Antibody Staining Solution #2 (Cycle 1)
Blocking Buffer (Cycle 1)See aboveTop up to volume after adding antibodies
CD4 AF488R&D SystemsFAB8165G1:10
Ag85BAbcamab43019/NA1:100
Secondary Antibody Staining Solution #1 (Cycle 1)
Blocking Buffer (Cycle 1)See aboveTop up to volume after adding antibodies
Donkey Anti-Goat AF Plus 488Thermo Fisher ScientificA32814/AB_27628381:100
Donkey anti-Rabbit AF Plus 555Thermo Fisher ScientificA32794/AB_27628341:100
Donkey anti-Mouse AF Plus 647Thermo Fisher ScientificA32787/AB_27628301:50
Primary Antibody Staining Solution #3 (Cycle 1)
Blocking Buffer (Cycle 1)See aboveTop up to volume after adding antibodies
CD68 iF594Caprico Biotechnologies10641351:10
a-SMA DL755Novus BiologicalsNBP2-345221R/NA1:30
BSA-free Blocking Buffer (Cycle 2) 
Triton-X-100Sigma-AldrichT87870.3% by volume
Human BD Fc BlockBD Biosciences5642201% by volume
1x PBS Gibco10010-02398.7% by volume
Primary Antibody Staining Solution #4 (Cycle 2)
BSA-free Blocking Buffer (Cycle 2)See aboveTop up to volume after adding antibodies
CD15BD Biosciences3474201:30
Lumican BiotinR&D SystemsBAF28461:100
CD45 PENovus BiologicalsNBP2-34528PE1:20
CD20 eFluor 660Thermo Fisher Scientific50-0202-821:10
Pan-Cytokeratin AF750Novus BiologicalsNBP2-33200AF7501:10
Secondary Antibody Staining Solution #2 (Cycle 2)
BSA-free Blocking Buffer (Cycle 2)See aboveTop up to volume after adding antibodies
Donkey anti-Mouse IgM AF488Jackson ImmunoResearch715-545-020/AB_23408441:100
Strepavidin AF594Thermo Fisher ScientificS112271:200

References

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Multiplex ImagingAutofluorescent SamplesSpatial ProteomicsFFPE Lung TissuePhotoirradiation ProtocolAntibody Panel DesignWhole Slide ImagingGranulomatous InflammationImmune Cell Mapping
Optimized Workflow for Iterative Bleaching Extends Multiplexity Imaging of Highly Autofluorescent Clinical Samples
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