Name: multiplexed fluorescent immunohistochemical staining, imaging, and analysis in histological samples of lymphoma
Uploaded: 2019-01-09T14:00:00
Description: Watch this Scientific Journal Video about Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma at JoVE.com

S.-B.N. and A.D.J. are supported by the Singapore Ministry of Health&#39;s National Medical Research Council Transition Awards (NMRC/TA/0020/2013 and NMRC/TA/0052/2016). The authors acknowledge a Yong Siew Yoon Research Grant to A.D.J. from the National University Cancer Institute of Singapore toward the purchase of a Vectra spectral imaging microscope. This study is approved by the Singapore NHG Domain Specific Review Board B (2014/00693).

All tissues used in this protocol were obtained under the Singapore NHG Domain Specific Review Board B study 2014/00693.
1. Selection and Validation of Antibodies
NOTE: Before proceeding with the establishment of any multiplexed panel, ensure that all antibodies stain robustly, identifying only the target antigen of interest. The aim is to select antibodies that specifically recognize the antigen of interest in tissue sections.
<ol>
	<li>For an antibody with a wel...

<ol>
	<li>Swerdlow, S. H., 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=.">.</a> WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. , (2017).</li><li>Swerdlow, S. H., 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+2016+revision+of+the+World+Health+Organization+classification+of+lymphoid+neoplasms.">The 2016 revision of the World Health Organization classification of lymphoid neoplasms.</a> Blood. 127 (20), 2375-2390 (2016).</li><li>Dabbs, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Diagnostic Immunohistochemistry. Theranostic and Genomic Applications. , (2010).</li><li>Kim, S. -. W., Roh, J., Park, C. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+for+Pathologists:+Protocols,+Pitfalls,+and+Tips.">Immunohistochemistry for Pathologists: Protocols, Pitfalls, and Tips.</a> Journal of Pathology and Translational Medicine. 50 (6), 411-418 (2016).</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>Anderson, M. W., Natkunam, Y., Jones, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+profiling+of+lymphoma.">Immunohistochemical profiling of lymphoma.</a> Neoplastic Hematopathology: Experimental and Clinical Approaches. , 21-44 (2010).</li><li>Parra, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Platforms+of+Multiplexed+Immunofluorescence+for+Study+of+Paraffin+Tumor+Tissues.">Novel Platforms of Multiplexed Immunofluorescence for Study of Paraffin Tumor Tissues.</a> Journal of Cancer Treatment and Diagnosis. 2 (1), 43-53 (2018).</li><li>Cregger, M., Berger, A. J., Rimm, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+and+Quantitative+Analysis+of+Protein+Expression.">Immunohistochemistry and Quantitative Analysis of Protein Expression.</a> Archives of Pathology &amp; Laboratory Medicine. 130 (7), 1026-1030 (2006).</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>T&#243;th, Z. E., Mezey, &. #. 2. 0. 1. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Visualization+of+Multiple+Antigens+with+Tyramide+Signal+Amplification+using+Antibodies+from+the+same+Species.">Simultaneous Visualization of Multiple Antigens with Tyramide Signal Amplification using Antibodies from the same Species.</a> Journal of Histochemistry &amp; Cytochemistry. 55 (6), 545-554 (2007).</li><li>Ng, S. -. B., 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+Analysis+of+a+Multiplexed+Immunofluorescence+Panel+in+T-Cell+Lymphoma.">Quantitative Analysis of a Multiplexed Immunofluorescence Panel in T-Cell Lymphoma.</a> SLAS TECHNOLOGY: Translating Life Sciences Innovation. 23 (3), 252-258 (2017).</li><li>Bogusz, A. M., 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=Diffuse+large+B-cell+lymphoma+with+concurrent+high+MYC+and+BCL2+expression+shows+evidence+of+active+B-cell+receptor+signaling+by+quantitative+immunofluorescence.">Diffuse large B-cell lymphoma with concurrent high MYC and BCL2 expression shows evidence of active B-cell receptor signaling by quantitative immunofluorescence.</a> PLoS One. 12 (2), e0172364 (2017).</li><li>Kalyuzhny, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Signal Transduction Immunohistochemistry: Methods and Protocols. , (2011).</li><li>Bordeaux, 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=Antibody+validation.">Antibody validation.</a> BioTechniques. 48 (3), 197-209 (2010).</li><li>PerkinElmer. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Vectra 3 Quantitative Pathology Imaging System User's Manual. , (2012).</li><li>Ho, J., Tumkaya, T., Aryal, S., Choi, H., Claridge-Chang, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+beyond+P+values:+Everyday+data+analysis+with+estimation+plots.">Moving beyond P values: Everyday data analysis with estimation plots.</a> bioRxiv. , (2018).</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+T+and+B+Cells+in+Murine+Spleen+and+Tumor.">Multispectral Imaging of T and B Cells in Murine Spleen and Tumor.</a> The Journal of Immunology Author Choice. 196 (9), 3943-3950 (2016).</li><li>PerkinElmer. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Opal Multiplex IHC Assay Development Guide. , (2017).</li></ol>

mf-IHC has the potential to enable pathologists to refine diagnosticcriteria in lymphoid pathology and to analyze the role of biomarkers in specific cell types toward a prediction of clinical outcome. As a new research method, mf-IHC is increasingly applied to the quantitative and spatial identification of multiple immune parameters of tumor cells17. The detection of mf-IHC for the co-expression of tumor biomarkers has been shown to be reproducible and reliable5. However, t...

Lymphoid neoplasms are caused by the uncontrolled malignant proliferation of lymphocytes. These cells are vital components of the immune system and localize to the primary and secondary immune organs, such as the bone marrow, lymph nodes, spleen, and other mucosa-associated lymphoid system. Lymphoid neoplasms are a heterogeneous group of disorders who are classified based on a constellation of features, including morphology, immunophenotype, genetic features, and clinical presentation. While each parameter plays a part, lineage remains a defining feature and forms the basis for the WHO classification system which recognizes neoplasms derived from B cells, T cells, and natural killer (NK) cells1.
Key to the classification of lymphoma has been the characterization of the antibodies against leukocyte surface markers of the various subtypes of lymphocytes2. Immunohistochemistry (IHC) has been traditionally used for the analysis of such markers and is based on the principle of the specific antigen-antibody recognition to detect cell- and tissue-based molecules that can be visualized through the light microscope3. However, the identification of multiple targets on a single slide by conventional bright-field chromogenic multiplex IHC has limitations because it is often difficult to distinguish multiple color signals on a single tissue section reliably&#8212;especially for antigens with a very low expression4. Visual assessment and quantification of staining can also be subjective, causing variability in the analysis and data interpretation5.
Therefore, conventional IHC on formalin-fixed, paraffin-embedded (FFPE) samples is not feasible for the simultaneous detection of multiple targets in immunologically diverse diseases like lymphoma. Furthermore, distinguishing neoplastic lymphocytes from the surrounding immune cells is often imprecise. This hinders studies looking at the relevance of novel biomarkers in lymphoma. In this context, multiplex fluorescent IHC (mf-IHC) offers a promising alternative as it allows the quantitative assessment of antigen coexpression and a spatial relationship with higher precision while conserving limited samples6,7. When this imaging technology is partnered with the digital image analysis software, the data interpretation is made more efficient and facilitates the study of tumor and microenvironment heterogeneity8,9. In this protocol, a tyramide-based immunofluorescence (IF) multiplexing method is applied to amplify the signal and is compatible with any IHC-validated antibody from any host species, even those developed in the&#160;same species5,7,10. The tyramide-based protocol allows for the direct conjugation of the fluorophore to the tissue of interest so that the primary and secondary antibody can be stripped after each step, allowing for the sequential application of multiple stains without antibody cross-reactivity.
A multiplexed strategy will be useful for predicting prognosis and treatment outcomes by identifying targets and their variant immunologic patterns in lymphomas. Multiplex fluorescent IHC has been applied in our lab for the study of a panel of T and B lymphocyte markers and T-follicular helper markers in angioimmunoblastic T-cell lymphoma (AITL), a subtype of a peripheral T-cell lymphoma characterized by aggressive clinical behavior and tumor heterogeneity11. The utility of this method is also illustrated in diffuse large B-cell lymphoma (DLBCL) where the increased signaling of a B-cell receptor with simultaneous C-MYC and BCL-2 expression suggests the potential therapeutic use of Bruton&#39;s tyrosine kinase inhibition12. 
Here we describe the entire protocol from antibody validation to the selection of appropriate control tissues and multiplexing using lymphoma FFPE tissues, with an eventual analysis of stained slides using a scanning automated quantitative pathology imaging system.

<table>
	<tbody>
		<tr>
			<td>Antibody diluent</td>
			<td>DAKO</td>
			<td>REF S3022</td>
			<td>Blocking</td>
		</tr>
		<tr>
			<td>Peroxidase Blocking Solution</td>
			<td>DAKO</td>
			<td>S2023</td>
			<td>For peroxide blocking</td>
		</tr>
		<tr>
			<td>Vectra multispectral automated microscope</td>
			<td>Perkin Elmer</td>
			<td>Vectra2.0.8</td>
			<td>Spectral imaging</td>
		</tr>
		<tr>
			<td>absolute Ethanol</td>
			<td>EMSURE</td>
			<td>1.00983.2500</td>
			<td>Ethyl alcohol for rehydration</td>
		</tr>
		<tr>
			<td>Amplification Diluent</td>
			<td>PERKIN ELMER</td>
			<td>FP1135</td>
			<td>Fluorophore diluent buffer</td>
		</tr>
		<tr>
			<td>Anti-Mouse IgG [Goat] HRP-Labeled</td>
			<td>PERKIN ELMER</td>
			<td>NEF822001EA</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Anti-Rabbit IgG [Goat] HRP-Labeled</td>
			<td>PERKIN ELMER</td>
			<td>NEF812001EA</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>BCL2</td>
			<td>DAKO</td>
			<td>clone 124 ( Lot No. 20031561)(RRID-AB578693)</td>
			<td>primary antibody</td>
		</tr>
		<tr>
			<td>BCL6</td>
			<td>LEICA</td>
			<td>NCL-L-Bcl6-564(Lot No.6050438)(RRID-AB563429)</td>
			<td>primary antibody</td>
		</tr>
		<tr>
			<td>CD20</td>
			<td>DAKO</td>
			<td>Clone L26 (Lot No.20028627) (RRID-AB442055)</td>
			<td>primary antibody</td>
		</tr>
		<tr>
			<td>c-MYC</td>
			<td>ABCAM</td>
			<td>Y 69 clone ab32072 (Lot NO.GR29511133)(RRID-AB731658)</td>
			<td>primary antibody</td>
		</tr>
		<tr>
			<td>Cy 5</td>
			<td>PERKIN ELMER</td>
			<td>FP1171</td>
			<td>Appropriate tyramide based fluorescent reagent</td>
		</tr>
		<tr>
			<td>Graphpad Prism 7</td>
			<td>Graph pad</td>
			<td></td>
			<td>Statisitcal software</td>
		</tr>
		<tr>
			<td>HistoClear Clearing Agent</td>
			<td>SIGMA</td>
			<td>H2779-1L</td>
			<td>Histoclear for dewaxing and clearing</td>
		</tr>
		<tr>
			<td>inForm Advanced 
			Image Analysis Software</td>
			<td>Perkin Elmer</td>
			<td>Inform Software 2.2.1</td>
			<td>Data Analysis software</td>
		</tr>
		<tr>
			<td>KI67</td>
			<td>DAKO</td>
			<td>Clone MIB-1 (Lot No.20040401) (RRID-AB2314699)</td>
			<td>primary antibody</td>
		</tr>
		<tr>
			<td>KOS MILESTONE multifunctional tissue processor</td>
			<td>Milestone</td>
			<td></td>
			<td>Microwave for Heat induced epitope retrieval</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>PANASONIC</td>
			<td>NN-ST651M</td>
			<td>Microwave stripping</td>
		</tr>
		<tr>
			<td>Mowiol</td>
			<td>SIGMA ALDRICH</td>
			<td>81381 Aldrich Mowiol&#174; 4-88</td>
			<td>mounting media</td>
		</tr>
		<tr>
			<td>Opal 570</td>
			<td>PERKIN ELMER</td>
			<td>FP1488</td>
			<td>Appropriate tyramide based fluorescent reagent</td>
		</tr>
		<tr>
			<td>Opal520</td>
			<td>PERKIN ELMER</td>
			<td>FP1487B21</td>
			<td>Appropriate tyramide based fluorescent reagent</td>
		</tr>
		<tr>
			<td>Opal540</td>
			<td>PERKIN ELMER</td>
			<td>FP1494</td>
			<td>Appropriate tyramide based fluorescent reagent</td>
		</tr>
		<tr>
			<td>Opal620</td>
			<td>PERKIN ELMER</td>
			<td>FP1495</td>
			<td>Appropriate tyramide based fluorescent reagent</td>
		</tr>
		<tr>
			<td>Poly-L-lysine coated slide</td>
			<td>FISHER SCIENTIFIC</td>
			<td>120-550-15</td>
			<td>Slide for tissue section adhesion in routine histological use</td>
		</tr>
		<tr>
			<td>Spectral DAPI</td>
			<td>PERKIN ELMER</td>
			<td>FP1490</td>
			<td>nucleic acid stain</td>
		</tr>
		<tr>
			<td>Target Retrieval Solution, pH9.0(10x)</td>
			<td>DAKO</td>
			<td>S2367</td>
			<td>For HIER</td>
		</tr>
		<tr>
			<td>Tris Buffer saline (TBS)</td>
			<td>1st BASE</td>
			<td>BUF3030 20X4L</td>
			<td>for buffer wash</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>SIGMA ALDRICH</td>
			<td>P1379-1L</td>
			<td>Tween</td>
		</tr>
	</tbody>
</table>

multiplexed fluorescent immunohistochemical staining, imaging, and analysis in histological samples of lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

mf-IHC images for a DLBCL sample with C-MYC and BCL2 gene rearrangement (double-hit lymphoma) are shown in Figure 1. Figure 2 illustrates the simulated bright-field immunohistochemical images. Figure 3 indicates the generation of percentage data. Figure 4 displays the details of a median formula for the generation of numeric data. Figure 5 s...

Watch this Scientific Journal Video about Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma at JoVE.com

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

Lymphomas are histologically complex tumors. Therefore, multiplexed fluorescent immunohistochemistry offers advantages over conventional chromogenic immunohistochemistry to study markers of interest in the tumor cells and the microenvironment. This technique has the potential for standardizing scoring of antibody based staining in histological lymphoma samples, a process that has historically been challenging due to the complexity of the tumor microenvironment.Specifically, multiplexed fluorescent immunohistochemistry and spectral imaging allows the quantitative measurement of multiple stains within a single slide, thereby allowing the assessment of antigen co expression and spacial relationship within clinical material. Begin by immersing the de-waxed and rehydrated slides in a standard antigen retrieval buffer in a microwave save glass jar and performing heat-induced epitope retrieval in a suitable microwave. Perform additional rounds of microwave stripping based on the position of the antibody in the final multiplex sequence.For example, the fourth antibody requires three additional rounds of microwave stripping prior to staining. After completion of the stripping process, use forceps and heat-proof gloves to transfer the slides to distilled water at room temperature for an at least 10 minute cool down. When the antigen retrieval solution has cooled, block the tissue peroxidase activity with a commercial peroxidase block for 10 minutes followed by a five minute wash in tris-buffered saline, or TBS, and a non ionic detergent.Block any nonspecific staining with a 10 minute incubation in blocking buffer followed by incubation with the primary antibody of interest at room temperature for 30 minutes and three five minute washes with TBS-D buffer. Next, incubate the samples with an appropriate horseradish peroxidase conjugated secondary antibody for 15 minutes at room temperature, followed by three washes in TBS-D buffer as demonstrated. After the last wash, apply an appropriate tyramide based fluorescent reagent to the slides for a five minute incubation at room temperature followed by three five minute TBS-D washes.Perform an additional microwave based stripping to remove the primary and secondary antibodies in fresh antigen retrieval solution as demonstrated. At the end of the retrieval, cool the slides in distilled water and dry the areas on the slides without tissue with lab wipes. Incubate slide with dappy for 10 minute at room temperature followed by three TBS-D washes.Then, carefully dry the slides with lab wipes without contacting the tissues and mount the samples with an appropriate mounting medium for imaging. For monoplex slide scanning, select the appropriate filters from the fluorophores used for labeling the samples and examine each marker and its corresponding fluorescence channel to identify a suitable exposure time for obtaining a clean signal, focusing on the tissue component that should have the strongest signal for the marker. To allow cross-sample comparison of the pixel intensity, use the live camera setting to adjust the exposure time in each individual channel until there are no overexposed areas in the live camera image.When all of the slides have been scanned, acquire simulated bright field images to visually compare with the normal immunohistochemical pattern and to determine if the staining pattern is correct. To scan tissue microarray slides, use the tissue microarray scanning mode in the imaging system and an appropriate auto focus algorithm. For whole tissue section slides, have the slides reviewed by a qualified pathologist to select optimal images from the most representative tumor areas.Before beginning the analysis, select a tumor marker to identify the cells of interest for the analysis. Have a pathologist review the images and decide whether tissue segmentation is required. If required, select the appropriate control regions for segmentation and check whether the image analysis software can correctly identify such regions.After the cell segmentation, have the pathologist review the segmentation map to ensure the fidelity of the intended segmentation approach and review individual images to determine whether the cell segmentation is adequate or if additional tissue segmentation is required to select regions enriched for tumor cells, stroma, and or necrosis. Then determine the optical intensity positive cutoff value for each marker in conjunction with the pathologist and generate histograms in an appropriate statistic software program to analyze the frequency distribution of the marker intensity per cell. To generate percentage data for specific markers of interest within defined cells, insert a pivot table into the datasheet and select Sum to calculate the positivity percentage of a single marker of interest.The total number of marker positive cells divided by the total number will be calculated. The result is the marker positive cell percentage with one core, or one study number. To generate numeric data, obtain the mean intensity of each marker of interest in all of the cells study within a sample to extract the median normalized count for each marker of each core or study number.Here, representative multiplex fluorescent immunohistochemical images for a diffuse large B-cell lymphoma sample with c-MYC and BCL2 gene rearrangement are shown. Simulated bright field immunohistochemical images demonstrate a similar tumor marker staining pattern. The application of multiplex fluorescent immunohistochemical images to a T-cell panel in angioimmunoblastic T-cell lymphomas reveals the cell heterogeneity of the tumor sample.Here, the optimization of a tonsil control sample and the resulting data analysis are shown. A variety of image analysis software programs can be used after the unmixing step to quantitate marker expression and localization in tumor cells and the tumor microenvironment. This technique paves the way for researchers to study the co expression of multiple markers within specific cell types in single tissues sections of lymphoma.Take care to prevent heat-induced injury during the microwaving steps and be aware of fall risks during the scanning steps that are performed in the deck.

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Research

JoVE Journal

Immunology and Infection

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to the best of these authors' knowledge, no method has been developed to allow for the in situ visualization of the 
bacteria in histological sections.
While bacterial localization has been determined in vivo in previous studies using plate counts 3, radiometric labeled 4, 
or bioluminescent bacteria 5, most of these studies have only been performed at the gross organ level, with one exception 6. This limitation 
is of particular concern because E. ictaluri has a complex infection cycle 1,7, and it has a variety of virulence factors 8,9. 
The complex interaction of E. ictaluri with its host is similar in many respects to Salmonella typhi 10, which is in the same taxonomic family.
Here we describe a technique allowing for the detection of bacteria using indirect immuno-histochemistry using the monoclonal Ed9 antibody described by Ainsworth et al.11.
Briefly, a blocking serum is applied to paraffin embedded histological sections to prevent non-specific biding. Then, the 
sections are incubated with the primary antibody: E. ictaluri specific monoclonal antibody Ed9. Excess antibodies are rinsed away and the FitC 
labeled secondary antibodies are added. After rinsing, the sections are mounted with a fluorescent specific mounting medium. 
This allowed for the detection of E. ictaluri in situ in histological sections of channel catfish tissues.

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

Here we describe a procedure allowing the labeling of Edwardsiella ictaluri in situ in histological sections from channel catfish Ictalurus punctatus using indirect immunohistochemistry with monoclonal antibodies Ed9 as a primary, and fluorescent FitC labeled antibodies as a secondary. This allowed for the detection of the bacterium using fluorescent microscopy.

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to ...

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of experimental models needs to be as rigorous as that applied for human samples. Indeed, drawing reliable and accurate conclusions is critically influenced by the quality of tissue section preparation. Here, we describe a protocol for histopathological analysis of murine tissues that implements several automated steps during the procedure, from the initial preparation to the paraffin embedding of the murine samples. The reduction of methodological variables through rigorous protocol standardization from automated procedures contributes to increased overall reliability of murine pathological analysis. Specifically, this protocol describes the utilization of automated processing and embedding robotic systems, routinely used for the tissue processing and paraffin embedding of human samples, to process murine specimens of intestinal inflammation. We conclude that the reliability of histopathological examination of murine tissues is significantly increased upon introduction of standardized and automated techniques.

Lack of standardization for murine tissue processing reduces the quality of murine histopathological analysis as compared to human specimens. Here, we present a protocol to perform histopathological examination of murine inflamed and uninflamed colonic tissues to show the feasibility of robotic systems routinely used for processing and embedding&#160;human samples.

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of ...

Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia selectively), when combined with IBA1 (which provides an exceptional visualization of their morphology), allows investigation of changes in density, distribution, and morphology. Quantifying these parameters is important in providing insights into the roles exerted by microglia, the resident macrophages of the brain. Under normal physiological conditions, microglia are regularly distributed in a mosaic-like pattern and present a small soma with ramified processes. Nevertheless, as a response to environmental factors (i.e., trauma, infection, disease, or injury), microglial density, distribution, and morphology are altered in various manners, depending on the insult. Additionally, the described double-staining method allows visualization of infiltrating macrophages in the brain based on their expression of IBA1 and without colocalization with TMEM119. This approach thus allows discrimination between microglia and infiltrating macrophages, which is required to provide functional insights into their distinct involvement in brain homeostasis across various contexts of health and disease. This protocol integrates the latest findings in neuroimmunology that pertain to the identification of selective markers. It also serves as a useful tool for both experienced neuroimmunologists and researchers seeking to integrate neuroimmunology into projects.

This protocol describes a step-by-step workflow for immunofluorescent costaining of IBA1 and TMEM119, in addition to analysis of microglial density, distribution, and morphology, as well as peripheral myeloid cell infiltration in mouse brain tissue.