Name: Iterative Bleaching Extends Multiplicity with Use of Staining Automation for Core Facilities (Video) | JoVE Journal
Uploaded: 2025-08-06T14:00:00.000+00:00
Duration: 4 min 52 s
Description: Learn the IBEX method for enhanced tissue imaging using automated stainers and tyramide signal amplification. This approach improves staining consistency and detection sensitivity in neuroscience research.

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Emily Speranza is supported by start-up funds from the Cleveland Clinic Foundation. This work utilized instruments acquired from NIH SIG grants (S10OD026983 &#38; S10OD030269 to N.A.C.).

&#8203;1. Building of IBEX panels with TSA-based dyes
NOTE: Always use a fresh preparation of antibodies and dyes and store at 4 &#176;C when not in use.
<ol>
	<li>Antigen retrieval sensitivity testing
	<ol>
		<li>Preparation of slides
		<ol>
			<li>Use a microtome to collect three serial sections for each antibody to test from the same FFPE-embedded tissue onto three separate, positively charged slides.</li>
			<li>Label slides with the antibody to be tested, along with the number of antigen retrieval cycles to be tested: 1 cycle, 3 cycles, and 6 cycles.</li>
			<li>Dry the slides overnight at room temperature. If working with surface markers only, bake slides at 60 oC for at least 20 min to soften the wax. If working with nuclear markers, bake the slides at 60 oC overnight.</li>
		</ol>
		</li>
		<li>Prep the program on the autostainer for the different number of cycles, ensuring there are three wash steps between each step. See Table 1 for example protocols for 1, 3, and 6 cycle tests. 
		NOTE: For each individual antibody, ensure a consistent time for the antibody step and the TSA dye application. 
		&#8203;For TSA-based dye application, an alternative to allowing the dye to sit for 3-10 min at room temperature is to allow the dye to incubate for 20 min at 37 &#176;C.</li>
		<li>Load the samples onto the autostainer and run the protocol for the associated number of cycles.</li>
		<li>Repeat this process from steps 1.1.1 to 1.1.3 for each individual antibody to be validated and tested.</li>
		<li>Remove the slides from the autostainer and place them in PBS.</li>
		<li>Mount the slides with aqueous mounting media (same as for an IBEX round) and a #1.5 coverslip for optimal imaging.</li>
		<li>Use an epifluorescence microscope to image the 1 cycle slide for the TSA dye and DAPI, making note of the exposure time needed to visualize the signal.</li>
		<li>Repeat this for the 3 cycle and 6 cycle slides, noting the exposure time needed to visualize the TSA dye.</li>
		<li>Compare exposure times across the different cycle numbers. See Figure 1 for examples. Ensure three wash cycles between all steps.
		<ol>
			<li>If the exposure time decreases with increased cycles, place this antibody and stain at a later step.</li>
			<li>If exposure time increases with increased cycles, place this antibody in an earlier step.</li>
			<li>If there is no large change in exposure time, place this antibody at any step.</li>
			<li>Group antibodies based on cycle requirements for the initial pass at IBEX panels. 
			NOTE: For the best results, perform this validation on all desired markers for a given IBEX panel. Perform this validation each time tissue types are changed or when working with polyclonal primary antibodies with unique lot numbers.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Address bleedthrough of signal from spectrally nearby dye by assessment of signal strength.
	<ol>
		<li>Group antibodies into potential IBEX panels based on the optimal number of retrieval cycles, as was determined from above into 1, 3, or 6 cycle groups.</li>
		<li>Preparation of slides
		<ol>
			<li>Use a microtome to create serial sections from an FFPE sample, one for each antibody to use in a single IBEX panel.</li>
			<li>Label each slide with one of the antibodies to be used in the IBEX panel.</li>
			<li>Let the slides dry overnight at room temperature. If working with surface markers only, let the slides bake at 60 oC for at least 20 min to soften the wax. If working with nuclear markers, let the slides bake at 60 oC overnight.</li>
		</ol>
		</li>
		<li>For the 1, 3, and 6 cycle group, use the one cycle setup from Table 1. Ensure three washes between all steps
		<ol>
			<li>Modify the protocol for each antibody to be used with the optimal staining time.</li>
			<li>Use the same TSA time for all the antibodies.</li>
		</ol>
		</li>
		<li>Stain the samples on the autostainer.</li>
		<li>Remove the samples and place the slides in PBS.</li>
		<li>Mount the slides with aqueous mounting media (same as for an IBEX round) and a #1.5 coverslip for optimal imaging.</li>
		<li>Image the slides on an epifluorescence microscope using optimal exposure time for each image.</li>
		<li>Determine signal strength. Slides with short exposure times have a very high signal strength, while slides with a long exposure time have a low signal strength.</li>
		<li>Use the signal strength along with the cycle number to develop IBEX panels.
		<ol>
			<li>Group antibodies based on cycle number.</li>
			<li>For a given group of antibodies with a specific cycle number, ensure that a strong signal is not optically close to a weak signal to minimize signal spillover.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Validate the individual IBEX panels to ensure each panel can generate the desired outcome.
	<ol>
		<li>Prepare the slides.
		<ol>
			<li>Use a microtome to create serial sections, one for each multiplex IBEX panel, and label with the IBEX panel order to be used.</li>
			<li>Let the slides dry overnight at room temperature. If working with surface markers only, let the slides bake at 60 oC for at least 20 min to soften the wax. If working with nuclear markers, let the slides bake at 60 oC overnight.</li>
		</ol>
		</li>
		<li>Set up the autostainer.
		<ol>
			<li>For the first panel, set up the autostainer program with no additional antigen retrieval steps.</li>
			<li>For the second panel, introduce additional denaturation steps before the first antibody equal to the number of denaturation steps in the first panel. See Table 2 for an example panel 1 with five antibodies, and Table 3 for an example panel 2 with four antibodies. Ensure three wash steps between all steps.</li>
			<li>Repeat as needed for additional IBEX panels, adding in more denaturation steps.</li>
		</ol>
		</li>
		<li>Remove the slides from the autostainer and place them in PBS until ready to mount.</li>
		<li>Mount the slides with aqueous mounting media (same as for an IBEX round) and a #1.5 coverslip for optimal imaging.</li>
		<li>Image the slides on an epifluorescence microscope using optimal exposure time for each image and each TSA dye.</li>
		<li>Assess if all panels are displaying properly. If so, move onto IBEX imaging. If not, re-address signal balancing and retrieval cycles. 
		NOTE: Some adjustments can be made within a single panel to optimize signal and prevent the &#34;umbrella effect&#34;. Some highly abundant signals can block staining of downstream signals. Look within a given panel to use more sparsely present stains first (see Figure 2).</li>
	</ol>
	</li>
</ol>
2. IBEX adaptation with autostainers using TSA reagents and whole slide imaging platforms (Figure 3)
NOTE: For comparison of the two validated autostainers used and discussed in this protocol, see Table 4.
<ol>
	<li>Prepare all slides for the IBEX run.
	<ol>
		<li>Use a microtome to create FFPE sections on a positively charged slide. Be sure to include one slide for each experimental sample, positive control slides of tissue sections known to contain the antigens to be imaged, and a negative control slide in which PBS is used in place of all primary antibodies. 
		NOTE: If working in batches, include a batch control sample. A batch control sample is a single FFPE tissue block that is sectioned and included in each batch of experimental samples for use in normalization and control of experimental variation between batches of staining samples. This is only necessary if not all samples will be processed together.</li>
		<li>Let the slides dry overnight at room temperature. If working with surface markers only, let the slides bake at 60 oC for at least 20 min to soften the wax. If working with nuclear markers, let the slides bake at 60 oC overnight.</li>
	</ol>
	</li>
	<li>Prepare antibodies and dyes for the first IBEX panel to be imaged.</li>
	<li>Set-up the autostaining program. See Table 2 for an example of a 5-marker panel. Ensure three washes between each step.</li>
	<li>Load samples and antibodies into the autostainer and run the program to stain the samples.</li>
	<li>Remove the slides from the autostainer and apply a coverslip using aqueous mounting media appropriate for multispectral imaging and a #1.5 coverslip.</li>
	<li>Image the slides within 2 days of staining</li>
	<li>Fluorescence Imaging on epifluorescence or fluorescence slide scanner
	<ol>
		<li>Starting with the well-controlled positive control sample, load the positive control sample into the microscope.
		<ol>
			<li>Locate a positive signal in the sample for each of the different antigens to be imaged.</li>
			<li>Set the exposure time to ensure capture of the dynamic range of the stain while minimizing background by starting at a low exposure and gradually increasing the exposure time until the signal is present. Do this for each dye individually. 
			NOTE: Test for the presence of both high and low expression of the markers to ensure capture of the full dynamic range.</li>
		</ol>
		</li>
		<li>Save the settings used for the positive control sample and use them for all further imaging steps.</li>
		<li>Load and image the negative control sample and all the experimental samples using the settings determined by the positive control sample.</li>
		<li>Verify that the images are of optimal quality before proceeding to the subsequent steps.</li>
	</ol>
	</li>
	<li>Coverslip removal
	<ol>
		<li>Immerse slides in a Coplin jar with DI water. Place the Coplin jar in a water bath set to 65 &#176;C for 15-20 minutes to facilitate natural removal of the&#160;coverslip. Alternatively, if an aqueous-based mounting medium is used or the mounting medium has hardened completely, remove coverslips by soaking in phosphate-buffered saline (PBS) or DI water overnight to allow coverslips to naturally fall off.</li>
	</ol>
	</li>
	<li>Bleaching with LiBH4
	<ol>
		<li>In a fume hood, prepare a fresh solution of LiBH4 at a concentration of 1mg/mL in DI water. Make enough to cover each sample 3x over. 
		NOTE: Do not prepare more than 10 mL at a time due to the reaction of LiBH4 with water. Use DI water and not Millipore/nanopure water for optimal bleaching activity.</li>
		<li>Let the solution rest for 20 min. Bubbles will form.</li>
		<li>Pipette 1 mL of LiBH4 solution onto each slide in a staining chamber in a fume hood. Let the solution rest on the slide for 10-15 min.</li>
		<li>Remove the LiBH4 by pipetting off the slide and replace with a fresh 1 mL of LiBH4. Let the solution rest for 10-15 min.</li>
		<li>Remove the LiBH4 by pipetting off the slide and replace with a fresh 1 mL of LiBH4. Let the solution rest for 10-15 min.</li>
		<li>Remove the LiBH4 from the slides by pipetting and submerge the slides in DI water or PBS for short-term storage (no more than 1 day).</li>
		<li>Within a day, move onto the next IBEX staining round.</li>
		<li>Optional: wet mount slides and perform a quick visualization on an epifluorescence scope to verify bleaching success.</li>
	</ol>
	</li>
	<li>Wet mount of slide into autostainer and second IBEX staining run
	<ol>
		<li>Prepare all antibodies and TSA dye solutions for the autostainer.</li>
		<li>For the Ventana autostainer:
		<ol>
			<li>When ready to start autostaining, pipette approximately 1 mL of autostainer wash buffer onto the slide to prevent drying out of the wet mount.</li>
			<li>Load wet-mounted slides into the autostainer.</li>
		</ol>
		</li>
		<li>For the Bond Rx:
		<ol>
			<li>Place cover tiles on slides.</li>
			<li>At the top of the covertile, pipette 300 &#181;L of wash buffer to allow the solution to flow into the space between the sample and the covertile.</li>
			<li>Load samples onto the autostainer.</li>
		</ol>
		</li>
		<li>Set up the autostainer program following the example in Table 2 with N set to the number of antibodies in the panel.</li>
		<li>Remove the slides from the autostainer and apply a coverslip using aqueous mounting media appropriate for multispectral imaging and a #1.5 coverslip.</li>
		<li>Image the slides within 2 days of staining following the sample procedure described in step 2.7 for setting up the imaging session and collecting images.</li>
		<li>After imaging, bleach samples as described in steps 2.8-2.9.</li>
	</ol>
	</li>
	<li>Repeat for all IBEX panels desired.</li>
</ol>
3. Analysis of samples with commercially available software
<ol>
	<li>Complete whole slide scans of the stained slides before proceeding to alignment.</li>
	<li>If the software allows, perform spectral unmixing through the automated unmixing function built into the scanning functions, including removal of the autofluorescence spectra. If this is a function of the software being used, select the unmixing option and run using standard settings and pre-set dyes 
	NOTE: Custom unmixing can be generated in most software that allows for unmixing. To create a library, individual tissue sections stained with a single fluorophore are added to the software&#39;s measurements. A region of unstained or autofluorescent tissue is also scanned to characterize the autofluorescence spectrum, and this is selected with the autofluorescence tool. These spectra are compiled within the program by hitting &#34;prepare image&#34; and then exported to a project-specific unmixing library. This library is then selected as the unmixing library in the unmixing option.</li>
	<li>Import the scanned slides into commercially available image analysis software for alignment.</li>
	<li>Slide registration of multiple IBEX rounds
	<ol>
		<li>Select two iterations of IBEX rounds to be registered.</li>
		<li>Right click on the two and select the Register option.</li>
		<li>Select the Default Registration with High-resolution Registration for an initial attempt at registration.</li>
		<li>Alternatively, select Single Channel Registration from the options and select a single channel that was consistent across all the IBEX runs. 
		NOTE: This option is useful for dyes that are resistant to bleaching (see materials for which dyes do not bleach).</li>
	</ol>
	</li>
	<li>Image alignment
	<ol>
		<li>Open one of the registered images.</li>
		<li>In the Tools tab, under Image Registration, select Registration Tools to open all registered images.</li>
		<li>In the Registration Tools tab, select the landmark function.</li>
		<li>Use the landmark function to fine tune the alignment of whole slides.
		<ol>
			<li>Mark landmarks on one slide image manually.</li>
			<li>On the other IBEX images, mark the same landmark. The program will refine the alignment using the identified landmark. Repeat for fine tuning of alignment.</li>
		</ol>
		</li>
		<li>Once satisfied with the alignment, fuse the slides using the Fuse | Serial Stain function.</li>
		<li>Repeat this process with any additional IBEX imaging rounds until a final composite of all the images is completed.</li>
	</ol>
	</li>
	<li>Image analysis
	<ol>
		<li>Segmentation of cells
		<ol>
			<li>Under the Analysis tab, select and open the HighPlex module, then select the phenotyping module for cell identification.
			<ol>
				<li>Load all the markers and start by finding all the nuclei using the Nuclear Detection tab in the Highplex module. Load markers in the Dye Selection tab. Identify cells by using the DAPI stain.</li>
				<li>In the Analyze tab, turn on the Real-Time Tuning tool using the following key parameters: Minimum Nuclear Intensity set to filter out background signal; Maximum Image Brightness set to control overall signal visibility; Nuclear Segmentation Aggressiveness set to refine the boundaries of detected nuclei.</li>
				<li>Tune each parameter carefully to ensure accurate segmentation.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Detect the cytoplasm and membrane markers.
		<ol>
			<li>Navigate to the Membrane and Cytoplasm Detection tab.</li>
			<li>Set the Minimum Cytoplasm Radius to a value appropriate for the sample for the expected size of the cells.</li>
			<li>Under the Membrane Dyes option, specify which channels are membrane markers. 
			NOTE: This should result in better segmentation of individual cells for further analysis.</li>
		</ol>
		</li>
		<li>Marker thresholding and phenotype assignment
		<ol>
			<li>For each marker, set the thresholds for positive versus negative signals. Do this under the tabs generated for each marker.</li>
			<li>Do the manual thresholding for each marker to identify populations.</li>
			<li>Alternatively, use a bioinformatics-based automated approach to thresholding. In the Advanced | Store Object (Cell) Data, toggle the option to True. In Export | Object Data, export the dataset to get marker expression information for each identified cell.</li>
			<li>Import these data into additional software for further and tailored analysis. Options for software for further analysis are found in Table 5.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol><li>Radtke, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IBEX%3A+an+iterative+immunolabeling+and+chemical+bleaching+method+for+high-content+imaging+of+diverse+tissues%5BTitle%5D)+AND+%22Nature+Protoc%22%5BJournal%5D)">IBEX: an iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues.</a> Nature Protoc. 17 (2), 378-401 (2022).</li>
<li>Radtke, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IBEX%3A+A+versatile+multiplex+optical+imaging+approach+for+deep+phenotyping+and+spatial+analysis+of+cells+in+complex+tissues%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">IBEX: A versatile multiplex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues.</a> Proc Natl Acad Sci U S A. 117 (52), 33455-33465 (2020).</li>
<li>Prichard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overview+of+automated+immunohistochemistry%5BTitle%5D)+AND+%22Arch+Pathol+Lab+Med%22%5BJournal%5D)">Overview of automated immunohistochemistry.</a> Arch Pathol Lab Med. 138, 1578-1582 (2014).</li>
<li>Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplexed+immunohistochemistry%2C+imaging%2C+and+quantitation%3A+a+review%2C+with+an+assessment+of+Tyramide+signal+amplification%2C+multispectral+imaging+and+multiplex+analysis%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis.</a> Methods. 70, 46-58 (2014).</li>
<li>Speranza, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-related+differences+in+immune+dynamics+during+SARS-CoV-2+infection+in+rhesus+macaques%5BTitle%5D)+AND+%22Life+Sci+Alliance%22%5BJournal%5D)">Age-related differences in immune dynamics during SARS-CoV-2 infection in rhesus macaques.</a> Life Sci Alliance. 5, 4(2022).</li>
<li>Parra, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Procedural+requirements+and+recommendations+for+multiplex+immunofluorescence+tyramide+signal+amplification+assays+to+support+translational+oncology+studies%5BTitle%5D)+AND+%22Cancers+%28Basel%29%22%5BJournal%5D)">Procedural requirements and recommendations for multiplex immunofluorescence tyramide signal amplification assays to support translational oncology studies.</a> Cancers (Basel). 12 (2), 255(2020).</li>
<li>Hoyt, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplex+immunofluorescence+and+multispectral+imaging%3A+forming+the+Basis+of+a+clinical+test+platform+for+immuno-oncology%5BTitle%5D)+AND+%22Front+Mol+Biosci%22%5BJournal%5D)">Multiplex immunofluorescence and multispectral imaging: forming the Basis of a clinical test platform for immuno-oncology.</a> Front Mol Biosci. 8, 674747(2021).</li>
<li>Bankhead, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((QuPath%3A+Open+source+software+for+digital+pathology+image+analysis%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">QuPath: Open source software for digital pathology image analysis.</a> Sci Rep. 7, 16878(2017).</li>
<li>Feng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial+analysis+with+SPIAT+and+spaSim+to+characterize+and+simulate+tissue+microenvironments%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Spatial analysis with SPIAT and spaSim to characterize and simulate tissue microenvironments.</a> Nat Commun. 14, 2697(2023).</li>
<li>Meyer, L., Eling, N., Bodenmiller, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((cytoviewer%3A+an+R%2FBioconductor+package+for+interactive+visualization+and+exploration+of+highly+multiplexed+imaging+data%5BTitle%5D)+AND+%22BMC+Bioinformatics%22%5BJournal%5D)">cytoviewer: an R/Bioconductor package for interactive visualization and exploration of highly multiplexed imaging data.</a> BMC Bioinformatics. 25, 9(2024).</li>
<li>Windhager, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+end-to-end+workflow+for+multiplexed+image+processing+and+analysis%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">An end-to-end workflow for multiplexed image processing and analysis.</a> Nat Protoc. 18 (11), 3565-3613 (2023).</li>
</ol>

The authors have no conflicts of interest to declare.

Here, we provide considerations for adapting the IBEX protocol into core pathology facilities with the application of automated staining equipment and optimization of TSA-based reagents in the IBEX protocol. We demonstrate how to best optimize for the repeated rounds of antigen retrieval and proper loading in round 2 or higher onto the automated equipment. In addition, we demonstrate an additional method of performing alignment using the Halo software that may be available in core facilities. These approaches have been demonstrated to be useful in determining the differences in lung environments in non-human primate tissue following SARS-CoV-2 infections5. 
A key component of this work is highlighting the need for extensive optimization of the IBEX panel design prior to staining of experimental tissues. In particular, with the use of TSA-based reagents, some considerations not seen in the base IBEX methods must be taken into consideration. The first is the impact of multiple rounds of antigen retrieval on antibody sensitivity. With TSA-based reagents, each new antibody development is preceded by antibody denaturation. Some antibodies/antigen pairs will be susceptible to additional rounds of antigen retrieval and diminish assay sensitivity, whereas some will obtain enhanced sensitivity with repetitive denaturation/antigen retrieval. Careful testing of each antibody in the target tissue before designing the panels is important to ensure best staining practices and inform the sequence order of both the antibodies and fluorescent dyes. Buffers, including both citrate-based (pH 6.0) and Tris-EDTA-based (pH 9.0), are tested for all antibodies to determine the optimal antigen retrieval conditions. This involves systematically evaluating each buffer to identify which one maximizes antigen exposure for a given antibody. Additionally, temperature (e.g., 20-37 &#176;C), incubation time (typically 30-60 min), and blocking conditions are optimized to reduce non-specific background staining and ensure specific binding of the primary and secondary antibodies, if necessary. Blocking before the primary and secondary antibodies may include the use of commercially available blocking reagents to account for tissue-specific autofluorescence or non-specific interactions. Other important considerations that must be optimized before finalizing IBEX panels include the umbrella effect, where shielding of the epitopes can occur if the order is not catered to the specific research question being asked6. To avoid fluorescent bleedthrough, it is typical to put higher-expressing antigen targets into far red channels that have the least bleedthrough and weaker markers in the 480-520 nm range, which will have the best signal-to-noise ratio given these fluorescent dyes are the brightest7. However, care should be given to highly autoflourescent tissue samples that may be brightest in this range; we recommend addressing this issue by imaging tissue without any dyes to address potential levels of autoflouresence. Together, these optimization steps, taken with all normal optimization and controls typically done with IBEX, can inform proper panel design for IBEX with TSA reagents. 
In this method, we have demonstrated the use of two common autostainers. However, these approaches could be applied to other autostainer methods as well. The key part here is the optimal removal of coverslips and the wet load for additional staining rounds. Additionally, we do not specify any specific microscope setup. Though kits may recommend the use with certain imaging systems for optimal slide scanning, any microscope setup, particularly an epifluorescence microscope with compatible filter cubes and a broad-spectrum LED light source, should work. Some dyes require multispectral detectors or specialized filter cubes, which may not be available in all laboratories and can present challenges when used with standard filter cubes due to their spectral overlap. These dyes are better separated using multispectral imaging systems or highly specific filters. Some fluorophores fall outside the range of conventional far-red filters (typically ~700 nm) and require a specialized filter cube or detector capable of capturing near-infrared signals. Specialized microscopes are built with these filters, but alternatives are available. These will require additional optimization to determine how the dyes perform in a given microscope configuration.
Multispectral unmixing is highly recommended for multiplex fluorescent IHC, particularly when working with tissues prone to autofluorescence, as it effectively separates the true signal from background noise. Autofluorescence, which often overlaps with the emission spectra of commonly used fluorophores (particularly in higher energy wavelength channels), can obscure or interfere with specific signals, reducing accuracy. Spectral unmixing mathematically disentangles overlapping emission spectra, isolating real fluorescent signals while "cleaning up" unwanted autofluorescence. Spectral unmixing is particularly valuable in IBEX workflows, where achieving the cleanest possible signals is critical due to the iterative nature of the staining and imaging process. In IBEX, signals from multiple rounds of staining are imaged, bleached, and stacked on top of one another to create a composite image. Any autofluorescence or bleedthrough that remains unresolved in one round can propagate through the stacked images, complicating downstream analysis and reducing confidence in marker localization. Spectral unmixing effectively isolates true fluorescence signals from background autofluorescence, ensuring cleaner, more accurate signal detection in each individual round. This signal clarity is essential for generating reliable composite images where multiple markers are analyzed together across rounds, enabling confident phenotyping and spatial analysis. The imaging platform used for this protocol has onboard unmixing capabilities, but more unmixing options are needed for compatibility with other fluorescent whole-slide imaging platforms. 
In addition, a limitation of this work is the proprietary nature of the autostainers, as their associated reagents and wash buffers are optimized for these systems, but the specific formulations remain undisclosed. Furthermore, we have focused exclusively on one commercially available image analysis software for whole-slide image analysis and acknowledge that the cost of software licenses may present a financial barrier for some laboratories. Additional analysis software8,9 and options in R seem promising; in particular, the Bodenmiller Group10, who has created a package set that seems optimal for cores, in that IMCDataAnalysis11 is intended for reproducible workflow on multiplexed image downstream analysis in R. However, future efforts are needed to develop and characterize accessible and cost-effective alternatives for whole slide IBEX analysis to ensure broader adoption across resource-limited settings. Furthermore, this approach with the use of the autostainers for IBEX can be adapted for more traditional staining in IBEX, including with fluorescent conjugated primary antibodies, or with standard secondary antibodies without the TSA-based dyes if the tissue is of high enough quality and optimal fixation time to allow for good signal. 
Overall, we hope that this work will assist with the application of TSA-based multiplex fluorescent IHC using routinely utilized pathology core facility equipment to more widely adapt IBEX into these environments. This type of application truly highlights the versatility of the IBEX protocol and demonstrates its usefulness in even difficult tissue samples or in large batch approaches.

Iterative Bleaching Extends Multiplicity (IBEX) is a significant advancement in highly sensitive TSA-based, multi-round fluorescent immunohistochemistry, allowing for detailed visualization and quantification of numerous biomolecules within a single tissue sample. By employing repeated cycles of bleaching and re-staining, IBEX enables the examination of complex cellular environments with unparalleled depth and clarity, making it a powerful tool in the field of immunostaining and multiplexed imaging1,2. 
Many academic, governmental, and industry entities host core pathology facilities dedicated to supporting a diverse array of tissue-based research methodologies aimed at generating qualitative and quantitative morphomolecular datasets. These cores are critical resources, providing specialized tissue-based services that allow research labs to outsource complex and labor-intensive molecular pathology processes and consult directly with pathologists in study design, assay development, and data interpretation. To ensure consistency and reduce variability across experiments, these core facilities routinely rely on commercially available autostainers-automated instruments designed to perform standardized staining protocols with high reproducibility and efficiency3. 
While autostainers have become the backbone of immunostaining in core facilities, enabling high-throughput and consistent results, they have not yet been adapted to accommodate more recent advancements like IBEX. Furthermore, the need for specialized knowledge in panel design, particularly when using tyramide signal amplification (TSA)-based reagents4, adds an additional layer of complexity, limiting the broader adoption of IBEX in routine research workflows. As a result, researchers who wish to take advantage of IBEX's high-resolution, multiplexed imaging capabilities may often be limited by their core facilities' existing equipment. This gap between innovative imaging techniques and the practical realities of core facility operations highlights a barrier to the broader adoption of IBEX in academic research. This work aims to address this barrier by demonstrating the successful application of IBEX on common autostainer platforms, focusing on the use of TSA-based reagents using formalin-fixed, paraffin-embedded (FFPE) tissues. We first discuss the steps necessary to build an IBEX panel using TSA-based dyes, and the cover application of these panels to two different commercially available autostainers. Together, this offers practical guidance on antibody-antigen retrieval sensitivity testing, discussing key considerations for optimizing panel design, and providing an alternative method for panel alignment using commercial software that may be available and through core pathology laboratories.

<table><tbody><tr><td>Staining reagent&lt;br/&gt; Alexa TSA kits</td><td>ThermoFisher</td><td></td><td>Alternative TSA dyes if Opal microscopy filters are not available. These include Alexa Fluor 488 (blue/green channel), Alexa Fluor 555 or 568 (red channel), Alexa Fluor 647 (far-red channel), Alexa Fluor 750 (near-infrared, if equipped with appropriate filters).&amp;nbsp;</td></tr><tr><td>Antibody&lt;br/&gt; Arg1</td><td>Cell Signaling</td><td>93668</td><td>Used in Figure 4 at 1:100</td></tr><tr><td>Material&lt;br/&gt; Blades</td><td>Leica Biosystems</td><td></td><td>Or Similar, high or low profile will depend on tissue type</td></tr><tr><td>Antibody&lt;br/&gt; CD11b</td><td>Abcam</td><td>ab133357</td><td>Used in Figure 4 at 1:2000</td></tr><tr><td>Antibody&lt;br/&gt; CD163</td><td>Abcam</td><td>ab182422</td><td>Used in Figure 4 at 1:500</td></tr><tr><td>Antibody&lt;br/&gt; CD31</td><td>Cell Signaling</td><td>77699S</td><td>Used in Figure 2 at 1:100</td></tr><tr><td>Antibody&lt;br/&gt; CD3e</td><td>Dako</td><td>A045201-2</td><td>Used in Figure 1 at 1:100</td></tr><tr><td>Tool&lt;br/&gt; Coplin Jar</td><td>Fischer Scientific</td><td>01-816-21</td><td>Or Similar</td></tr><tr><td>Material&lt;br/&gt; Coverslips</td><td>VWR</td><td>v48393-026</td><td>Or Similar</td></tr><tr><td>Antibody&lt;br/&gt; CX3CR1</td><td>Abcam</td><td>ab308613</td><td>Used in Figure 4 at 1:450</td></tr><tr><td>Stain&lt;br/&gt; DAPI</td><td>Akoya</td><td>FP1490</td><td></td></tr><tr><td>Software&lt;br/&gt; FIJI</td><td></td><td></td><td>Used to measure signal intensity</td></tr><tr><td>Slide&lt;br/&gt; Fisher Superfront Slides</td><td>Fisher Scientific</td><td>22-037-246</td><td>Or similar</td></tr><tr><td>Software&lt;br/&gt; HALO</td><td>Indica Labs</td><td></td><td>Example commerically available image analysis software. Used to align and analyze images.</td></tr><tr><td>Antibody&lt;br/&gt; Iba1</td><td>Cell Signaling</td><td>17191</td><td>Used in Figure 4 at 1:800</td></tr><tr><td>Software&lt;br/&gt; inForm</td><td>Akoya</td><td></td><td>Used to measure signal intensity</td></tr><tr><td>Antibody&lt;br/&gt; iNOS</td><td>Abcam</td><td>ab283655</td><td>Used in Figure 4 at 1:400</td></tr><tr><td>Instrument&lt;br/&gt; Leica Bond Rx (or RXm)</td><td>Leica Biosystems</td><td>21.2201</td><td>Referred to as &quot;Autostainer 1&quot; in text. Ensure all associated reagents are also avaiable including staining boxes and bulk solutions.&amp;nbsp;</td></tr><tr><td>Reagent&lt;br/&gt; LiBH4</td><td>Sigma-Aldrich</td><td>62460-5G-F</td><td>Keep in an anhydrous environment</td></tr><tr><td>Instrument&lt;br/&gt; Mantra Snap</td><td>Akoya</td><td></td><td>Multi-spectral flourscent microscope is needed to deteced the dyes. Example microscope that can be used to detect Opal dyes.</td></tr><tr><td>Tool&lt;br/&gt; Microtome</td><td>Leica Biosystems</td><td></td><td>Or Similar with prefered accessories such as brushes</td></tr><tr><td>Staining reagent&lt;br/&gt; Opal IHC 480 Kit</td><td>Akoya</td><td>FP1500001KT</td><td>Used in Figure 4</td></tr><tr><td>Staining reagent&lt;br/&gt; Opal IHC 6-plex Plex Kit</td><td>Akoya</td><td>NEL821001KT</td><td>520 and 620 do not bleach, used in Figure 1, 2, and 4</td></tr><tr><td>Staining reagent&lt;br/&gt; Opal IHC 780 Kit</td><td>Akoya</td><td>FP1501001KT</td><td>Used in Figure 4</td></tr><tr><td>Tool&lt;br/&gt; Oven</td><td>Millipore Sigma</td><td>Z683329</td><td>Or Similar, can also be done on autostainer</td></tr><tr><td>Antibody&lt;br/&gt; Pancytokeratin</td><td>Abcam</td><td>ab9377</td><td>Used in Figure 4 at 1:50</td></tr><tr><td>Reagent&lt;br/&gt; PBS (optional)</td><td>Fisher Scientific</td><td>AAJ61196AP</td><td></td></tr><tr><td>Instrument&lt;br/&gt; PhenoImager</td><td>Akoya</td><td></td><td>Multispectral whole slide imaging with onboard spectral unmixing included in software</td></tr><tr><td>Reagent&lt;br/&gt; ProLong Gold Antifade</td><td>Fisher Scientific</td><td>P36961</td><td>Or similar aqueous mounting media</td></tr><tr><td>Antibody&lt;br/&gt; ProSP-C n-terminal (ProSPC)</td><td>Seven Hills Bioreagents</td><td>WRAB-9337</td><td>Used in Figure 4 at 1:250</td></tr><tr><td>Antibody&lt;br/&gt; RAGE</td><td>R&amp;amp;D</td><td>MAB1179</td><td>Used in Figure 1 and 2 at 1:50</td></tr><tr><td>Reagent&lt;br/&gt; Reaction buffer</td><td>Roche</td><td>NC1895692</td><td>For use with Ventana autostainer</td></tr><tr><td>Antibody&lt;br/&gt; SARS-CoV-2-N</td><td>Cell Signaling</td><td>68344</td><td>Used in Figure 2 at 1:1000</td></tr><tr><td>Slide&lt;br/&gt; TOMO Adhesion CC Microscope Slides-WH</td><td>Avantik</td><td>SKUSL6519-1</td><td>If tissue adherence is of concern</td></tr><tr><td>Instrument&lt;br/&gt; Ventana Discovery Ultra</td><td>Roche</td><td></td><td>Referred to as &quot;Autostainer 2&quot; in text. Ensure all associated reagents are also avaiable including staining boxes and bulk solutions.&amp;nbsp;</td></tr><tr><td>Reagent&lt;br/&gt; Wash buffer</td><td>Leica Biosystems</td><td>AR9590</td><td>For use with Leica Bond autostainer</td></tr><tr><td>Reagent&lt;br/&gt; Water</td><td>Fisher Scientific</td><td>15-230-147</td><td>DI is best, not millipore</td></tr><tr><td>Tool&lt;br/&gt; Water Bath</td><td>Leica Biosystems</td><td>140607020C1</td><td>Or Similar</td></tr></tbody></table>

iterative bleaching extends multiplicity with use of staining automation for core facilities

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

Iterative Bleaching Extends Multiplicity (IBEX) is a highly sensitive tyramide signal amplification (TSA)-based multi-round fluorescence imaging technique enabling detailed examination of numerous protein biomolecules in a single tissue sample through repeated bleaching, re-staining, and image co-registration. Despite its growing adoption for generating highly multiplexed immunostaining, implementing IBEX in an automated fashion through deployment in core facilities offers numerous benefits, including increased throughput, reproducibility, and overall efficiency of laboratory staff. Here, we demonstrate the successful application of IBEX on two broadly utilized autostainer platforms and discuss important optimization considerations for panel design using TSA-based reactions for formalin-fixed, paraffin-embedded (FFPE) tissue samples. This includes the methodology for testing antibody-antigen retrieval sensitivity, an important consideration for IBEX panel design when determining the sequence of antibody development. Additionally, we showcase downstream imaging approaches utilizing commercially available whole-slide scanners and illustrate quality alignment and analysis using commercially available cell phenotyping software. Through detailed descriptions of these methodologies and accompanying videos, we aim to highlight the versatility and ease of IBEX, offering practical guidance for core pathology facilities to integrate IBEX into their existing automated staining platforms.

We provide an example of an IBEX panel using TSA-based reagents on Autostainer 2 (Figure 4). Each marker can be clearly delineated, and there is minimal bleedthrough between markers. The image was collected across two rounds of staining. The sample is lung tissue following infection with Mycobacterium tuberculosis. The two IBEX staining panels were aligned using example commercially available software. Using the IBEX approach with optimized TSA reagents (as is described here), the spatial organization of immune cells, inflammatory markers, and specialized lung cells can be seen and appreciated. Proper optimization allows for markers to easily appear with minimal background.
<img alt="Immunofluorescence analysis of RAGE, CD3e in mouse lung, monkey spleen; antigen retrieval study." class="xfigimg" src="/files/ftp_upload/67853/67853fig1.jpg" data-altupdatedat="1783396841000" data-alt="Figure 1"> 
Figure 1: Antigen retrieval testing with a fluorophore with an emission of 520 nm of two different antigens. RAGE and CD3e were imaged utilizing conserved exposure times. The epitope of this RAGE antibody is highly sensitive and susceptible to repetitive antigen retrieval with rapid drop off of assay sensitivity with subsequent antigen retrievals. Conversely, CD3e assay sensitivity improves with multiple rounds of antigen retrieval. Scale bars = 50 µm. Abbreviation: RAGE = Receptor for Advanced Glycation Endproducts. <a href="https://www.jove.com/files/ftp_upload/67853/67853fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Immunofluorescence microscopy images showing RAGE and SARS-CoV-2 N protein interaction, cell staining." class="xfigimg" src="/files/ftp_upload/67853/67853fig2.jpg" data-altupdatedat="1783396841000" data-alt="Figure 2"> 
Figure 2: Example of the importance of sequence development with respect to the umbrella effect. RAGE is a highly expressed marker in AT1 cells, and in combination with CD31, is effectively blocking SARS-CoV-2 immunoreactivity. However, when SARS-CoV-2 IHC is performed first, the signals can all be visualized. Scale bars = 25 µm. Abbreviations: RAGE = Receptor for Advanced Glycation Endproducts; AT1 = Alveolar Type 1; SARS-CoV-2 = Severe acute respiratory syndrome coronavirus 2; IHC = Immunohistochemistry. <a href="https://www.jove.com/files/ftp_upload/67853/67853fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Tissue sectioning and staining diagram; microscope imaging; automated stainer; LiBH4 bleaching." class="xfigimg" src="/files/ftp_upload/67853/67853fig3.jpg" data-altupdatedat="1783396841000" data-alt="Figure 3"> 
Figure 3: Overview of the process using automated staining equipment for the IBEX protocol. Wet mounting for round 2 or higher of the staining process to prevent tissue from drying out. Abbreviation: IBEX = Iterative Bleaching Extends Multiplicity. <a href="https://www.jove.com/files/ftp_upload/67853/67853fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Multiplex immunofluorescence; diagram; markers DAPI, iNOS, CD11b; 480-780 nm spectral analysis." class="xfigimg" src="/files/ftp_upload/67853/67853fig4.jpg" data-altupdatedat="1783396841000" data-alt="Figure 4"> 
Figure 4: Example of IBEX with TSA-based dyes from autostainer 2 on a lung infected with Mycobacterium tuberculosis. Panel 1 contains all dyes, and panel 2 contains dyes that were amenable bleaching. A merged image of all channels across the full IBEX of the lung image is presented. Scale bars = 200 µm. Abbreviations: IBEX = iterative bleaching extends multiplicity; TSA = Tyramide Signal Amplification. <a href="https://www.jove.com/files/ftp_upload/67853/67853fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: An example protocol for validating effects of cycle number on antibodies for 1, 3, and 6 cycle tests. <a href="https://www.jove.com/files/ftp_upload/67853/67835_Table%201_17j.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: An example panel 1 with five antibodies for the IBEX autostainer for panel validation. <a href="https://www.jove.com/files/ftp_upload/67853/67835_Table%202_17j.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: An example panel 2 with four antibodies for the IBEX autostainer for panel validation. <a href="https://www.jove.com/files/ftp_upload/67853/67835_Table%203_17j.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: Comparison of two autostainers. Table summarizing the pros and cons of the Ventana Autostainer and the Bond Rx. <a href="https://www.jove.com/files/ftp_upload/67853/67853_Table%204.xlsx" target="_blank">Please click here to download this Table.</a>
Table 5: Software summary for image analysis. Table summarizing some of the analysis approaches and software tools available for the analysis of images post-segmentation. <a href="https://www.jove.com/files/ftp_upload/67853/67853_Table%205_17j.xlsx" target="_blank">Please click here to download this Table.</a>

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 (IBEX) is a highly sensitive tyramide signal amplification (TSA)-based multi-round fluorescence imaging technique ...

iterative-bleaching-extends-multiplicity-with-use-staining-automation

Research

JoVE Journal

Immunology and Infection

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

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

We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although the protocol was used to measure the mobility of yellow fluorescent protein-tagged p62 in aggresome-like induced structures, it can be applied to a variety of microscopy systems and fluorescent proteins.

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

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

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

This report describes an automated protocol for multiplex immunofluorescence (mIF) assays on an automated slide stainer. It demonstrates the high reproducibility of spatial proteomics on standard high-throughput tissue autostainers and whole-slide fluorescence imagers, which is ideal for custom mIF assays across a large number of tissue slides in translational research.

High-Throughput Automated Multiplex Immunofluorescence Assays for Translational Research

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

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

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.

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

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

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Learn the IBEX method for enhanced tissue imaging using automated stainers and tyramide signal amplification. This approach improves staining consistency and detection sensitivity in neuroscience research.

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

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