Editorial support was provided by Deborah Shuman of MedImmune.

NOTE: The protocol presented here describes how to perform immunoprofiling of an mIF panel by using TSA for six antibodies (CD68, ki67, PD-L1, PD-1, CD8, and AE1/AE3) on an automated stainer (see Table of Materials). The protocol also describes how to perform the drop controls for a quality control of a new mIF panel (see Supplemental Materials). In this protocol, staining is performed with eight unstained FFPE slides from human tonsil (positive control) and eight unstai...

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+pathologist-read+comparison+of+the+heterogeneity+of+programmed+death-ligand+1+(PD-L1)+expression+in+non-small+cell+lung+cancer.">Quantitative and pathologist-read comparison of the heterogeneity of programmed death-ligand 1 (PD-L1) expression in non-small cell lung cancer.</a> Modern Pathology. 30 (3), 340-349 (2017).</li><li>Dixon, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+multiplexing+techniques+for+immunohistochemistry.">Recent developments in multiplexing techniques for immunohistochemistry.</a> Expert Review of Molecular Diagnostics. 15 (9), 1171-1186 (2015).</li><li>Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+immunohistochemistry,+imaging,+and+quantitation:+a+review,+with+an+assessment+of+Tyramide+signal+amplification,+multispectral+imaging+and+multiplex+analysis.">Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis.</a> Methods. 70 (1), 46-58 (2014).</li><li>Feng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+immune+profiling+in+HPV-+oral+squamous+cell+cancer.">Multiparametric immune profiling in HPV- oral squamous cell cancer.</a> JCI Insight. 2 (14), (2017).</li><li>Feng, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+imaging+of+formalin-fixed+tissue+predicts+ability+to+generate+tumor-infiltrating+lymphocytes+from+melanoma.">Multispectral imaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma.</a> Journal for ImmunoTherapy of Cancer. 3, 47 (2015).</li><li>Granier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+immunofluorescence+analysis+and+quantification+of+intratumoral+PD-1++Tim-3++CD8++T+cells.">Multiplexed immunofluorescence analysis and quantification of intratumoral PD-1+ Tim-3+ CD8+ T cells.</a> Journal of Visualized Experiments. (132), e56606 (2018).</li><li>Parra, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+multiplex+immunofluorescence+panels+using+multispectral+microscopy+for+immune-profiling+of+formalin-fixed+and+paraffin-embedded+human+tumor+tissues.">Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues.</a> Scientific Reports. 7 (1), 13380 (2017).</li><li>Ribas, A., Wolchok, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+immunotherapy+using+checkpoint+blockade.">Cancer immunotherapy using checkpoint blockade.</a> Science. 359 (6382), 1350-1355 (2018).</li><li>Gorris, M. A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eight-color+multiplex+immunohistochemistry+for+simultaneous+detection+of+multiple+immune+checkpoint+molecules+within+the+tumor+microenvironment.">Eight-color multiplex immunohistochemistry for simultaneous detection of multiple immune checkpoint molecules within the tumor microenvironment.</a> Journal of Immunology. 200 (1), 347-354 (2018).</li><li>Parra, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+neoadjuvant+chemotherapy+on+the+immune+microenvironment+in+non-small+cell+lung+carcinomas+as+determined+by+multiplex+immunofluorescence+and+image+analysis+approaches.">Effect of neoadjuvant chemotherapy on the immune microenvironment in non-small cell lung carcinomas as determined by multiplex immunofluorescence and image analysis approaches.</a> Journal for ImmunoTherapy of Cancer. 6 (1), 48 (2018).</li><li>Rimm, D., Schalper, K., Pusztai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unvalidated+antibodies+and+misleading+results.">Unvalidated antibodies and misleading results.</a> Breast Cancer Research and Treatment. 147 (2), 457-458 (2014).</li><li>Baskin, D. G., Hewitt, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+state+of+the+science+of+immunohistochemistry:+the+Histochemical+Society's+standards+of+practice.">Improving the state of the science of immunohistochemistry: the Histochemical Society's standards of practice.</a> Journal of Histochemistry &amp; Cytochemistry. 62 (10), 691-692 (2014).</li><li>Freedman, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+need+for+improved+education+and+training+in+research+antibody+usage+and+validation+practices.">The need for improved education and training in research antibody usage and validation practices.</a> Biotechniques. 61 (1), 16-18 (2016).</li><li>Hewitt, S. 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This work was supported by MedImmune, the global biologics R&amp;D arm of AstraZeneca. C.W., K.R., and C.C.H. are employees of Perkin Elmer, which produces the reagents (TSA kit) and multispectral scanners that were used in this work. M.S., K.D., A.H., W.Z., C. Bagnall, C. Brown, J.C., A.L., K.S., M.R., and J.R.C. are employees of MedImmune with stock ownership and/or stock interests or options in AstraZeneca.

The ongoing cancer immunotherapy revolution is opening novel and promising therapeutic options for cancer patients13. Advances in the field of immuno-oncology will require increased knowledge of the inflammatory tumor microenvironment, not only to understand the biology of the immunological mechanisms involved in carcinogenesis but also to find predictive biomarkers for new immunotherapy-based treatments1,2. Due to the complex biology of c...

Immunoprofiling analysis of FFPE tumor tissue samples has become an essential component of immuno-oncology studies, particularly for the discovery and validation of novel predictive biomarkers for cancer immunotherapy in the context of clinical trials1,2. Chromogenic IHC, using chemical chromogens such as diaminobenzidine, remains the standard technique in diagnostic pathology for the immunolabeling of biopsy tissue3. Standard IHC can also be used for cancer tissue immunoprofiling, including the quantitation of subpopulations of tumor-associated lymphocytes and the assessment of expression levels of immune checkpoints such as programmed cell death ligand 1 (PD-L1)4,5. Standard IHC is limited, however, in that only one antigen can be labeled per tissue section. Because immunoprofiling studies typically require the analysis of the combined expression of several markers, the use of standard IHC would require the staining of multiple tissue sections, each stained with a single marker, and would, therefore, be substantially limited for the analysis of small tissue samples such as core needle biopsies. Standard IHC methods are also limited for the assessment of markers that are coexpressed by diverse cell populations, as is common with immune checkpoint markers such as PD-L1, which is expressed by both tumor-associated macrophages and cancer cells. This limitation has been reported in, for instance, the use of standard monoplex IHC by pathologists for the quantitative analysis of an IHC marker expressed by diverse cell types6. The development of multiplex chromogenic IHC techniques employing diverse colored chromogens on the same tissue section represents an advancement over the standard IHC monoplex method,7 although they remain limited by the immunolabeling of just a few markers and also present an important technical challenge for the proper evaluation of markers expressed in the same subcellular compartments of the same cell populations.
The aforementioned caveats regarding tissue availability from clinical samples, as well as the limitations of multiplex chromogenic IHC techniques, have given rise to the need to develop improved multiplex methods for immuno-oncology studies based on fluorescent labeling combined with imaging systems that can effectively separate the signals of multiple fluorophores from the same slide. One such technique is based on tyramide signal amplification (TSA) combined with multispectral microscopy imaging for efficient color separation8. A commercially available TSA-based kit employs fluorophores optimized for multispectral imaging8 (see Table of Materials). A critical advantage of this system is its compatibility with the same unlabeled primary antibodies that have already been validated and optimized for standard chromogenic IHC9,10,11. This allows not only faster optimization but also flexibility in the optimization and panel modifications incorporating new targets. Furthermore, the multiplex immunofluorescence (mIF) TSA method can be optimized for commercially available automated IHC stainer systems, allowing for a straightforward transfer from monoplex chromogenic IHC to mIF.
Here we present a protocol for an mIF panel for immuno-oncology studies that is based on automated mIF TSA staining and uses a multispectral scanner for imaging. This protocol can be adapted and modified by any laboratory user with access to the described instrumentation and reagents. The protocol includes a panel of six primary antibodies for the immunoprofiling of carcinomas: PD-L1, PD-1, CD68 (as a pan-macrophage marker), CD8 (T-cytotoxic cells), Ki-67, and AE1/AE3 (pan-cytokeratin, used as an epithelial marker for the identification of carcinoma cells). A recent study describes the optimization of a manual TSA mIF protocol by using chromogenic IHC as a standard reference to validate the multiplex staining12. The updated method presented here has been developed by using a commercially available, seven-color TSA kit optimized in an automated stainer, drastically shortening the staining time from 3&#8211;5 days to 14 h, while also improving the consistency of the staining. In addition to the detailed main protocol presented here, a Supplemental Materials section includes the &#34;drop-control&#34; method, an additional quality control process to evaluate a new mIF panel, as well as technical notes for the optimization, troubleshooting, and development of new multiplex panels to help the laboratory user to set up and optimize the mIF TSA method for customized mIF panels.

<table border="0" cellpadding="0" cellspacing="0" style="width:813px;" width="814">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">&#34;InForm 2.4.2&#34; Software for Spectral Unmixing and Image Analysis</td>
			<td style="width:123px;">PerkinElmer</td>
			<td style="width:119px;">CLS151066</td>
			<td style="width:299px;">Called &#34;spectral unmixing software&#34; in text</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:273px;">&#34;Phenochart 1.0.9&#34; QPTIFF Software for Selection of MSI and Overall Slide Scan Viewing</td>
			<td style="width:123px;">PerkinElmer</td>
			<td style="width:119px;">CLS151067</td>
			<td>Called &#34;QPTIFF software&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">#1.5 Coverslips</td>
			<td>Sigma Aldrich</td>
			<td>2975246</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200 Proof Ethanol</td>
			<td>Koptec</td>
			<td>V1001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20x Tris-Buffered Saline</td>
			<td>VWR</td>
			<td>J640-4L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Antibody Diluent</td>
			<td>DAKO</td>
			<td>S2203</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD68 Mouse Monoclonal</td>
			<td>DAKO</td>
			<td>M087601-2</td>
			<td>Clone PG-M1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD8 Rabbit Monoclonal</td>
			<td>Ventana</td>
			<td>M5392</td>
			<td>Clone SP239</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CK Mouse Monoclonal</td>
			<td>DAKO</td>
			<td>M351501-2</td>
			<td>Clone AE1/AE3</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-ki67 Mouse Monoclonal</td>
			<td>DAKO</td>
			<td>M724001-2</td>
			<td>Clone MIB-1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-PD-1 Rabbit Monoclonal</td>
			<td>Cell Signaling</td>
			<td>#86163</td>
			<td>Clone D4W2J</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-PD-L1 Rabbit Monoclonal</td>
			<td>Ventana</td>
			<td>790-4905</td>
			<td>Clone SP263</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Dewax Solution</td>
			<td>Leica</td>
			<td>AR9222</td>
			<td>Called &#34;dewax solution&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Epitope Retrieval Solution 1</td>
			<td>Leica</td>
			<td>AR9961</td>
			<td>Called &#34;ER1&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Epitope Retrieval Solution 2</td>
			<td>Leica</td>
			<td>AR9640</td>
			<td>Called &#34;ER2&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Open Containers, 30 mL</td>
			<td>Leica</td>
			<td>OP309700</td>
			<td>Called &#34;30 mL open containers&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Open Containers, 7 mL</td>
			<td>Leica</td>
			<td>OP79193</td>
			<td>Called &#34;7 mL open containers&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Bond Polymer Refine Detection</td>
			<td>Leica</td>
			<td>DS9800</td>
			<td>Called &#34;chromogenic detection kit&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Research Detection Kit</td>
			<td>Leica</td>
			<td>DS9455</td>
			<td>Called &#34;research detection kit&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Titration Kit</td>
			<td>Leica</td>
			<td>OPT9049</td>
			<td>Called &#34;titration kit&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Universal Covertile Novocastra</td>
			<td>Leica</td>
			<td>S21.2001</td>
			<td>Called &#34;covertiles&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Wash Solution 10X Concentrate</td>
			<td>Leica</td>
			<td>AR9590</td>
			<td>Called &#34;10x wash solution&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">BondRX Autostainer</td>
			<td>Leica</td>
			<td style="width:119px;"></td>
			<td>Called &#34;automated stainer&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">BondRX Software Version 5.2.1.204</td>
			<td>Leica</td>
			<td style="width:119px;"></td>
			<td>Called &#34;automated stainer software&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Opal 7-Color Automation IHC Kit</td>
			<td style="width:123px;">PerkinElmer</td>
			<td>NEL801001KT</td>
			<td style="width:299px;">Called &#34;multispectral staining kit&#34; in text</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peroxidase Block</td>
			<td>Leica</td>
			<td>RE7101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Diamond Antifade Mountant</td>
			<td>Thermo</td>
			<td>P36965</td>
			<td>Called &#34;slide mountant&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Starfrost Slides</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">15-183-51</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:273px;">Vectra Polaris Multispectral Microscope with &#34;Vectra 3.0.5&#34; Software for Multispectral Microscope Control</td>
			<td style="width:123px;">PerkinElmer</td>
			<td>CLS143455</td>
			<td>Called &#34;microscope control software&#34; in text</td>
		</tr>
	</tbody>
</table>

automated multiplex immunofluorescence panel for immuno-oncology studies on formalin-fixed carcinoma tissue specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4&#8242;,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

The protocol described here will provide results like those shown in Figure 2. Start with an evaluation of the staining in the tonsil control, beginning with the surface squamous cell epithelium. The histology of the tonsil sample can be reviewed with a pathologist, using the H&#38;E slide as a reference. If chromogenic IHC sections are performed with the same markers on the same tissue block, then these can be used to confirm the density and distribution of ...

Watch this Scientific Journal Video about Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens at JoVE.com

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

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.

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

This protocol is significant because the development optimization and transfer of multiplex immunofluorescence protocols is complex and challenging. For many labs the toe hold of an optimized protocol is invaluable. Multi-spectral immunofluorescence allows us to assess both the location and the identity of multiple cell types in and around the tumor.This is a powerful combination of information in immune oncology studies. The most compelling use for multiplex immunofluorescence is immune oncology. But the ability to survey multiple immune cell types in setu can be leveraged across all auto-immune and inflammatory therapies.Knowing the location and phenotype of immuno cells in and around solidual cells is both prognostic and predictable responses to tumor specific immunotherapy. The optimization and validation of the protocol are key steps that should not be rushed. New developers should familiarize themselves with the new kinds of artifacts associated with multi-spectral imaging.To locate the base multi-plex protocol preload it on the automated stainer. Filter for all'instead of preferred'on the protocol screen. Before making changes save the original protocol and click the protocols tab, and right click Opal 6 Multiplex to select copy.Change the name to 7 Multiplex Protocol 1'in the new window and select the tab associated with the correct device. Match the protocol in supplemental table S1'and click add reagent to add a ten minute paroxidies inhibition step, selecting the peroxidase inhibitor as the reagent. Confirm that the blocking steps utilize a protein kinase inhibitor blocking buffer, that each primary anti-body refers to the appropriate reagent, that the secondary anti-body steps utilize a horseradish peroxidase polymer and that the spectral dapi provided with the multi-spectral automation kit is utilized.Make sure that the protocol contains the peroxidase inhibitor, followed by six sequential staining runs each containing a protein blocking step, a primary anti-body, a secondary anti-body, a Tyramide floor, and then antigen retrieval to remove the anti-bodies. To add the drop controls, copy the 7 Multiplex Protocol 1'name, and change it to 7 Multiplex Protocol 1 CD68 Drop. Change the CD68 anti-body reagent to Mouse IgG and save the protocol.Then, create six more new protocols, one for each drop control, and one for the complete iso-type control in the same manner. Make a drop control protocol for each target in the same way as I've shown you here. To prepare a research detection kit, fill the 30 milliliter open container marked for 1X tris-buffered saline with 1X tris-buffered saline, and place the container in the position farthest from the handle in the Multiplex Research Kit 1.Label two 30 milliliter open containers muli-spectral block'and multi-spectral secondary, and fill the mutli-spectral block container with 30 milliliters of blocking buffer antibody diluent from the Multi-Spectral Staining Kit. Fill the multi-spectral secondary container with 30 milliliters of anti-Mouse anti-rabid secondary antibody polymer from the Multi-spectral Staining Kit and add 600 microliters of antibody diluent to each of ten 7 milliliter open containers. Next, add the concentrated primary antibody to the appropriate tubes in the volumes indicated in the table, and mix by gentle pipetting.Add 3 milliliters of double distilled water, plus 12 drops of spectral dapi into one 7 milliliter open container, and 3 milliliters of peroxidase block into a second 7 milliliter open container. Re-suspend each liathalized floor with 75 microliters of dimethyl sulfoxide and mix by pipetting. Then, store the Tyramide signal amplification floor stocks at 4 degrees Celsius until ready.While preparing the individual TSA floor dilutions. Register all reagents on the auto-stainer, then place each container into a reagent rack and load all of the reagents onto the automated stainer. The stainer will detect the presence and confirm the volume of each reagent.Once all of the reagents have been loaded, activate the protocol to run overnight. At the end of the staining procedure, mount the samples with cover slips and load the samples into the Multi-Spectral Imaging Platform Scanner for scanning. When all of the samples have been scanned, the images will be formatted as qptiff files.Open an appropriate qptiff analysis software program, and click load slide'to open a five filter whole slide scan of the slide of interest. Log in and use the stamp tool to select five regions for multi-spectral imaging. Under Select for:select Acquisition'under Size in Fields:select 1x1 and under Field resolution'select 0.5 micrometers 20x.Based on this cytokeratin staining select several regions with both cytokeratin positive tumor tissue, and cytokeratin negative stromal tissue to be imaged from the overall scan. When all of the multi spectral images have been selected, switch to the Vectra software and change the task from scan'to multi-spectral imaging'for each slide. Then, click scan to perform the multi-spectral imaging collection.When the multi-spectral image acquisition is complete, use the spectral un-mixing software to open files within the multi-spectral image folder. Under image format'select multi-spectral'Under sample format'select fluorescence'And under Spectral Library Source'select inForm'To select the floors, select and load all six floors, and dapi from the sample library slides, and click prepare all. The spectral un-mixing software will perform spectral unmixing on all of the open images using the provided spectral profiles.Identify and annotate the auto-fluorescent spectral profile. Be sure to check the auto-fluorescent feature to insure that it is completely captured in the auto-fluorescent channel after un-mixing and to test different samplings until it is removed acceptably. Then assess the staining pattern against the chromogenic single stains of the same target in sequential slides of the same tissue with a pathologist.To assess the fluorescence intensity of each channel, use the counts tool to survey the open images. Then, return to the staining protocol, and adjust the TSA concentration to address any normalized counts that exceed or fail to reach the acceptable range. In this representative analysis the tonsil tissue demonstrated a clearly defined cytokeratin staining within the tonsil surface, squamous epithelium without background in the lymphoid tissue with cytokeratin and PDL-1 staining apparent in the reticulated epithelium of the crypts.The follicular germinal centers were easily recognizable and rich in Ki-67 positive lymphocytes. The macrophages present within the germinal centers were identified by their CD-68 expression with some macrophages also observed outside of the germinal center. CD-8 stained lymphocytes with a T-cell distribution and PD-1 expression strongly stained small lymphocytes clustered at the periphery of the germinal center, as well as scattered throughout the interfollicular region.After the expected staining pattern was confirmed in the tonsil tissue control, the same staining could then be evaluated in a lung cancer sample. Isotype negative controls and drop controls should also be evaluated;not only for the detection of background and auto-fluorescence, but also for umbrella effects and spectral bleed. Drop controls are important for evaluating potential artifacts in the staining due to the multi-plex immunofluorescence method during the optimization of a new multi-plex panel.Each drop control should show the same staining pattern as the full multi-plex panel, except for the single primary antibody which should be removed on each specific control. Immuno profiling with multi-spectral immunofluorescence allows for the stratification of tumors and regions by immune criteria which can then be investigated by massively multiplex genomic, transcriptomic, and proteomic studies. I've used multi-plex immunofluorescence to investigate mechanisms of action in oncology and auto-immune disorders which allows for the identification and characterization of immune and pathogenic cells in a single section in setu.General precautions should be taken when working with anti-bodies against human proteins, Tyramide and dapi, but no reagent or material in this protocol is known to be particularly hazardous.

automated-multiplex-immunofluorescence-panel-for-immuno-oncology

Research

JoVE Journal

Cancer Research

20.2K Views.  MedImmune. Questo protocollo è significativo perché l'ottimizzazione dello sviluppo e il trasferimento di protocolli di immunofluorescenza multiplex è complesso e impegnativo. Per molti laboratori la presa di un protocollo ottimizzato è inestimabile. L'immunofluorescenza multispettrale ci consente di valutare sia la posizione che l'identità di più tipi di cellule all'interno e intorno al tumore.Questa è una potente combinazione di informazioni negli studi di oncologia immunitaria. L'uso pi...