This study was supported by Hong Kong Obstetrical and Gynecological Trust Fund in 2018 and Hong Kong Health and Medical Research Fund (06170186, 07180226).

The study was approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee. Informed consent was obtained from the participants before collecting the endometrial biopsies. Refer to the introduction section for inclusion criteria of the control and RM groups.
1. Sample preparation
<ol>
	<li>Ensure that all the women in this study undergo a daily urine dipstick test from day 9 of the menstrual cycle onwards to identify the luteinizing hormone (LH) surge to detect ovulation, and time the endometrial biopsies precisely on the 7th day after the LH surge (LH+7).</li>
	<li>Obtain a 0.5 cm2 fragment of endometrial biopsy using a Pipelle sampler or Pipet Curet from fertile women and women with unexplained RM. Label the container and place the specimen immediately in 10% neutral-buffered formalin (pH 7) for overnight fixation at room temperature. 
	NOTE: The volume of the fixative should be 5-10 times that of tissue.</li>
	<li>Place the tissue in a cartridge for dehydration in a tissue-processing machine before embedding it in molten paraffin wax. Embed the tissues in paraffin at 58-60 &#176;C.</li>
	<li>Allow the paraffin block to cool overnight at room temperature. Use a microtome to trim the paraffin blocks to a thickness of 3 &#181;m. 
	&#8203;NOTE: The use of distilled water aids in proper tissue mounting and adherence throughout multiplex staining.</li>
	<li>Place the paraffin ribbon in a water bath at 40-45 &#176;C for 30 s.</li>
	<li>Mount the sections onto poly-L-lysine (0.1% w/v)-coated microscope glass slides. Place the slides with the tissue facing upward and allow to dry at 37 &#176;C overnight. Store the slides in a slide box away from extreme temperatures until further use.</li>
</ol>
2. Determine the ideal concentration of antibodies for multiplex IHC using conventional IHC.
NOTE: This is important for identifying the expression level and pattern of each immune marker in the endometrium sample, and determine the staining sequence of each marker as well as their associated tyramide signal amplification (TSA) fluorophore pairings.
<ol>
	<li>Test the antibodies for their suitability for multiplex IHC by using manual conventional IHC13.</li>
	<li>Use endometrium tissues for single-antibody testing.</li>
	<li>Include a positive control (e.g., spleen) and negative control (isotype control) for optimizing the staining condition of each antibody.</li>
	<li>Perform the staining using the dilution recommended by the antibody&#39;s datasheet.</li>
	<li>Perform additional staining using concentrations above and below the recommended dilution used in step 2.3. 
	&#8203;NOTE: A clinical pathologist should assess the stained slides blindly to confirm the localization of the antibody probing and cellular integrity.</li>
</ol>
3. Multiplex staining method
<ol>
	<li>Slide preparation and fixation
	<ol>
		<li>Lay the slides (from step 1.6) flat with the tissue facing upwards in an oven and bake at 60 &#176;C for at least 1 h.</li>
		<li>Remove the slides from the oven and allow them to cool for at least 20 min at room temperature before placing them in a vertical slide rack.</li>
		<li>Dewax and rehydrate the formalin-fixed, paraffin-embedded slides with 10 min allotted to each of the following steps: xylene (2x), 100% ethanol (2x), 95% ethanol (1x), 70% ethanol (2x), and distilled water (2x).</li>
		<li>Place the rack of slides in a plastic slide box and submerge them in Tris-buffered saline (TBS, pH 7.6). 
		NOTE: The slides must remain moist starting from this rehydration step until mounting in the final step.</li>
		<li>Fix the samples by submerging the slides in a plastic slide box filled with a mixture of formaldehyde diluted in methanol (1:9) for 30 min in the dark.</li>
		<li>Wash the slides twice in deionized water for 2 min and then proceed to antigen retrieval.</li>
	</ol>
	</li>
	<li>Epitope retrieval
	<ol>
		<li>Place the rack of slides in a heat-resistant box and fill it with citric acid buffer (pH 6.0) to cover the slides.</li>
		<li>Place the box in the microwave, and heat the slides for 50 s at 100% power followed by 20 min at 20% power to maintain the same temperature.</li>
		<li>Allow the slides to cool for approximately 15 min at room temperature.</li>
		<li>Rinse the slides in water for 2 min followed by TBS-Tween 20 (TBST) for 2 min. 
		NOTE: TBST is composed of 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20 (v/v).</li>
	</ol>
	</li>
	<li>Blocking
	<ol>
		<li>Block endogenous peroxidase activity in the tissue by immersing the slide onto a jar containing peroxidase blocking solution (see the Table of Materials) for 10 min.</li>
		<li>Wash the slides with TBST for 5 min.</li>
		<li>Use a hydrophobic barrier pen to mark a boundary around the tissue section on the slide.</li>
		<li>Cover the tissue sections with a blocking buffer (see the Table of Materials) or bovine serum albumin (5%, w/v), and incubate the slides in a humidified chamber for 15 min.</li>
	</ol>
	</li>
	<li>Antibody and signal application
	<ol>
		<li>Remove the blocking reagents.</li>
		<li>Incubate with the primary antibody of interest (e.g., CD3, 1:100 dilution in antibody diluent, see Table 1) in a humidified chamber at room temperature for 30 min.</li>
		<li>Remove the primary antibody. Wash 3x with TBST for 5 min each time.</li>
		<li>Incubate with the polymer horseradish peroxidase (HRP)-labeled secondary antibody (Table 1) for 15 min in a humidified chamber at room temperature. Wash 3x with TBST for 5 min each time.</li>
		<li>Apply the Opal fluorophore TSA working solution (1:100 in amplification diluent) and incubate at room temperature for 10 min to allow fluorophore conjugation to the tissue sample at primary antibody binding sites. Wash with TBST in triplicate for 5 min each time.</li>
	</ol>
	</li>
	<li>Microwave-based stripping
	<ol>
		<li>Rinse with the antigen retrieval buffer (citrate buffer solution, pH 6.0).</li>
		<li>Perform microwave-based stripping to remove the primary-secondary-HRP complex to introduce the next primary antibody (e.g., CD20).</li>
		<li>Place the slides in antigen retrieval buffer, microwave them at 100% power for 50 s followed by 20% power for 20 min in microwave-safe containers, and cool them at room temperature for 15 min.</li>
		<li>Repeat steps 3.2.4 to 3.5.2 until the tissue samples have been probed with all the primary antibodies.</li>
	</ol>
	</li>
	<li>Counterstain and mounting
	<ol>
		<li>After microwave-based stripping and cooling of the antigen retrieval buffer, rinse the slides in distilled water and TBST.</li>
		<li>Incubate with 4&#39;, 6-diamidino-2-phenylindole (DAPI) solution (1.0 &#181;g/mL) for 5 min in a humidified chamber at room temperature.</li>
		<li>Wash 3x with TBST for 5 min each time. Wash with water once for 5 min.</li>
		<li>Air-dry the slide and mount with the appropriate mounting medium (see the Table of Materials). 
		NOTE: Counterstaining will not be required for monoplex slides to be used for spectral library development.</li>
	</ol>
	</li>
</ol>
4. Image and analysis
<ol>
	<li>Preparation of Spectral library slides
	<ol>
		<li>Create library slides (single-stain reference images) for each fluorophore, DAPI, and auto-fluorescence with the same control tissue for multispectral image analysis.</li>
		<li>Using the slides of the endometrial biopsy samples from women with RM and fertile women, perform steps 1.1 to 3.6.4 for single-antibody detection for each slide (without further antibody or fluorophore addition).</li>
		<li>For each antibody detection, stain one of the slides with DAPI (as in step 3.6.2) and leave one slide unstained for the detection of any possible tissue auto-fluorescence in the spectrum.</li>
		<li>Use the appropriate filters in the workstation to obtain the image for this set of slides for each antibody and upload them into the image analysis library (as described in step 4.2).</li>
		<li>Once the image is captured, select inForm as the Spectral Library Source&#160;and build the spectral library.</li>
		<li>As fluorophores are used, select Stains/Fluors... from the menu.</li>
		<li>While choosing the stains or fluorophores, narrow the choices by selecting one or more Groups. Select All to show all spectra compatible with the images.</li>
	</ol>
	</li>
	<li>Spectral imaging 
	NOTE: Images were captured using the Mantra Workstation with the spectral library established using the inForm Image Analysis software.
	<ol>
		<li>Capture the image of the single-antibody-stained slides with the appropriate epi-fluorescence filters as proposed in Table 2 (e.g., DAPI, fluorescein isothiocyanate [FITC], CY3, Texas Red, and CY5) using the workstation. 
		NOTE: Recommended filters for the specific fluorophores used in this protocol are shown in Table 2.</li>
		<li>Identify a suitable exposure time for an optimal signal by examining each marker in its corresponding fluorescence channel. 
		NOTE: The optimal signal is determined according to the reference on the positivity and localization obtained in the single antibody staining.</li>
		<li>Determine a fixed exposure time for each analyte (antibody-fluorophore combination) to standardize a cross-sample comparison. 
		NOTE: The determination of the fixed exposure is dependent on the intensity in the sample of interests.</li>
		<li>Scan the multiplex stained slides in the appropriate scanning mode with the embedded autofocus algorithm. 
		NOTE: The established spectral library would be used to differentiate the multispectral image cube into single individual components (spectral unmixing). This would allow the color-based identification for all markers of interest to be processed using the following two main steps: training session and image analysis session.</li>
	</ol>
	</li>
	<li>Image analysis
	<ol>
		<li>Cell counting of selected immune cell types in the endometrium</li>
		<li>Capture a minimum of 10 fields for analysis under 200x magnification. 
		NOTE: The fields were captured by scanning the whole section without any selection. This can ensure the simultaneous capture of all cell components of the endometrium, e.g., the luminal epithelial border, stroma, and glands.</li>
		<li>To count the cells, click on the Count Objects button in the step bar to display the Object Counting Settings panel.</li>
		<li>Check the Discard Object if Touching an Edge box for the exclusion of any objects touching the edge of the image, process region, or tissue region.</li>
		<li>If the tissue has been segmented, select the Tissue Category in which the objects are to be found. Do not count objects outside of the selected tissue category.</li>
		<li>Select the desired Approach to identify objects: Object-Based or Pixel-Based (Threshold). 
		NOTE: The Pixel-Based (Threshold) approach should be selected in case of a reliable or consistent stain for which the application of a simple threshold will yield the object pixels. The Object-Based approach is recommended when more advanced morphometry-based approaches are required in case of lack of consistency and specificity of staining of objects.</li>
		<li>Select the desired Signal Scaling: Auto Scale or Fixed Scale. 
		NOTE: Choosing Auto Scale will result in automatic scaling of each component plane before performing object segmentation. The Fixed Scale option is recommended when better segmentation performance is required, and stain signals are consistent and reliable.</li>
		<li>Select the Primary component for object segmentation from the drop-down list.</li>
		<li>Adjust the Minimum Signal value for the primary component to the desired threshold value.</li>
		<li>To automatically fill holes in objects, select Fill Holes.</li>
		<li>To detect objects that touch other objects as individual objects, instead of as one object, select the Refine Splitting check box after selecting the Maximum Size (pixels) check box.</li>
		<li>To exclude objects based on the roundness of the object, check the Roundness box and specify the desired Minimum Circularity.</li>
		<li>Count all stromal cells (CD3&#8722;/CD20&#8722;/CD68&#8722;/CD56&#8722;, and DAPI+), including the cells surrounding the blood vessels.</li>
		<li>Count the immune cells separately, including T cells (CD3+ and DAPI+), B cells (CD20+ and DAPI+), macrophages (CD68+ and DAPI+), and uNK cells (CD56+ and DAPI+).</li>
		<li>Express the data as the percentages of the immune cells relative to the total number of stromal cells for each captured image, and report the final cell count as an average of all fields.</li>
		<li>Use the View Editor to view the resulting data tables post processing. Export the Count Data table.</li>
		<li>Quantification of endometrial immune cell spatial distribution</li>
		<li>Under 200x magnification, estimate the L-function using the R program for a range of 0-20 &#181;m considered a cell-cell contact maximum distance14. 
		NOTE: The R language toolbox &#39;spatstat&#39; was used to measure the L-function.
		<ol>
			<li>Denote the level of clustering of different pairs of immune cells based on the area under the curve (AUC) of their L-function.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>ESHRE Guideline Group on RPL 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=ESHRE+guideline:+recurrent+pregnancy+loss.">ESHRE guideline: recurrent pregnancy loss.</a> Human Reproduction Open. 2018 (2), 004 (2018).</li><li>Stirrat, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+miscarriage.">Recurrent miscarriage.</a> Lancet. 336 (8716), 673-675 (1990).</li><li>Rai, R., Regan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+miscarriage.">Recurrent miscarriage.</a> Lancet. 368 (9535), 601-611 (2006).</li><li>Royal College of Obstetricians &amp; Gynaecologists. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+investigation+and+treatment+of+couples+with+recurrent+first-trimester+and+second-trimester+miscarriage.+Green-top+Guideline+No.+17.">The investigation and treatment of couples with recurrent first-trimester and second-trimester miscarriage. Green-top Guideline No. 17.</a> Royal College of Obstetricians &amp; Gynaecologists. , (2011).</li><li>King, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uterine+leukocytes+and+decidualization.">Uterine leukocytes and decidualization.</a> Human Reproduction Update. 6 (1), 28-36 (2000).</li><li>Le Bouteiller, P., Piccinni, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+NK+cells+in+pregnant+uterus:+why+there.">Human NK cells in pregnant uterus: why there.</a> American Journal of Reproductive Immunology. 59 (5), 401-406 (2008).</li><li>Lash, G. E., 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=Standardisation+of+uterine+natural+killer+(uNK)+cell+measurements+in+the+endometrium+of+women+with+recurrent+reproductive+failure.">Standardisation of uterine natural killer (uNK) cell measurements in the endometrium of women with recurrent reproductive failure.</a> Journal of Reproductive Immunology. 116, 50-59 (2016).</li><li>Yang, Y., 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=HOXA-10+and+E-cadherin+expression+in+the+endometrium+of+women+with+recurrent+implantation+failure+and+recurrent+miscarriage.">HOXA-10 and E-cadherin expression in the endometrium of women with recurrent implantation failure and recurrent miscarriage.</a> Fertility and Sterility. 107 (1), 136-143 (2017).</li><li>Tuckerman, E., Laird, S. M., Prakash, A., Li, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+value+of+the+measurement+of+uterine+natural+killer+cells+in+the+endometrium+of+women+with+recurrent+miscarriage.">Prognostic value of the measurement of uterine natural killer cells in the endometrium of women with recurrent miscarriage.</a> Human Reproduction. 22 (8), 2208-2213 (2007).</li><li>Jasper, M. 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=Macrophage-derived+LIF+and+IL1B+regulate+alpha(1,2)fucosyltransferase+2+(Fut2)+expression+in+mouse+uterine+epithelial+cells+during+early+pregnancy.">Macrophage-derived LIF and IL1B regulate alpha(1,2)fucosyltransferase 2 (Fut2) expression in mouse uterine epithelial cells during early pregnancy.</a> Biology of Reproduction. 84 (1), 179-188 (2011).</li><li>Kopcow, H. D., 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=T+cell+apoptosis+at+the+maternal-fetal+interface+in+early+human+pregnancy,+involvement+of+galectin-1.">T cell apoptosis at the maternal-fetal interface in early human pregnancy, involvement of galectin-1.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (47), 18472-18477 (2008).</li><li>Johnson, P. M., Christmas, S. E., Vince, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+aspects+of+implantation+and+implantation+failure.">Immunological aspects of implantation and implantation failure.</a> Human Reproduction. 14, 26-36 (1999).</li><li>Hong, G., 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+fluorescent+immunohistochemical+staining,+imaging,+and+analysis+in+histological+samples+of+lymphoma.">Multiplexed fluorescent immunohistochemical staining, imaging, and analysis in histological samples of lymphoma.</a> Journal of Visualized Experiments: JoVE. (143), e58711 (2019).</li><li>Carstens, J. L., 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=Spatial+computation+of+intratumoral+T+cells+correlates+with+survival+of+patients+with+pancreatic+cancer.">Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer.</a> Nature Communications. 8, 15095 (2017).</li><li>Zhao, Y., 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+use+of+multiplex+staining+to+measure+the+density+and+clustering+of+four+endometrial+immune+cells+around+the+implantation+period+in+women+with+recurrent+miscarriage:+comparison+with+fertile+controls.">The use of multiplex staining to measure the density and clustering of four endometrial immune cells around the implantation period in women with recurrent miscarriage: comparison with fertile controls.</a> Journal of Molecular Histology. 51 (5), 593-603 (2020).</li></ol>

The authors declare that they have no conflicts of interest to disclose.

Critical steps within the protocol 
It is important to note that multiplex staining requires diligent optimization. Antigen retrieval, using citrate buffer and microwave technology, requires optimization to ensure complete antibody stripping and maintain tissue viability. As TSA reagents covalently bind to sites surrounding the antigen, they can potentially inhibit the binding of a subsequent primary antibody through steric hindrance (also known as the &#34;umbrella effect&#34;). This tends to occur...

Recurrent miscarriage (RM) can be defined as the loss of two or more pregnancies before 24 weeks of gestation1. This frequent reproductive condition affects up to 1% of couples worldwide2,3. The pathophysiology is multifactorial and can be divided into embryologically driven causes (mainly due to an abnormal embryonic karyotype) and maternally driven causes that affect the endometrium and/or placental development. This manifestation can result from parental genetic abnormalities, uterine anomalies, prothrombotic conditions, endocrinology factors, and immunological disorders4.
In recent years, immune effector cell dysfunction has been implicated in the pathogenesis of early pregnancy loss5. This has inspired many investigations into elucidating the specific populations of immune cells in the endometrium during the menstrual cycle, implantation, and early pregnancy, with specific roles in early pregnancy. Among these immune cells, uterine natural killer (uNK) cells play a critical role during embryo implantation and pregnancy, particularly in the processes of trophoblastic invasion and angiogenesis6. Studies have shown an increased uNK cell density in the endometrium of women with RM7,8, although this finding was not associated with an increased risk of miscarriage9. However, this stimulated research evaluating the density of other immune cell types (such as macrophages, uterine dendritic cells) in the endometrium in women with RM10, 11. Nevertheless, it remains uncertain whether there is a significant alteration in the immune cell density in the peri-implantation endometrium in women with RM.
One possible explanation for the uncertainty is that evaluation of the endometrial immune cell density might be difficult due to the rapid changes in the endometrium during the window of implantation. During the 24 h timeframe, significant changes in the endometrium alter immune cell density and cytokine secretion, introducing a source of variation in these results12. In addition, most reports mainly rely on the use of single-cell staining (e.g., traditional IHC methods) that could not examine multiple markers on the same tissue section. Although flow cytometry can be used for detecting multiple cell populations in a single sample, the large amounts of cells required and the time-consuming optimization hinder the popularity and efficiency of this method. Hence, the recent advancement in multiplex IHC staining could solve this problem by immunostaining multiple markers on the same slide to evaluate multiple parameters, including cell lineage and histological localization of individual immune subpopulations. Further, this technology can maximize the information obtained in case of limited tissue availability. Ultimately, this technique can help elucidate the differences in immune cell interactions in the endometrium between fertile women and women with RM.
Two groups of women were recruited from the Prince of Wales Hospital, including fertile control women (FC) and women with unexplained recurrent miscarriage (RM). Fertile control was defined as women who had at least one live birth without any history of spontaneous miscarriage, and RM women were defined as those who had a history of &#8805;2 consecutive miscarriages before 20 weeks gestation. The subjects from the two groups met the following inclusion criteria: (a) age between 20 to 42 years old, (b) non-smoker, (c) regular menstrual cycle (25-35 days) and normal uterine structure, (d) no use of any hormonal regimen for at least 3 months before the endometrial biopsy, (e) no hydrosalpinx by hystero-salpingogram. In addition, all the subjects recruited had normal karyotyping, normal 3-dimensional ultrasonography hysterosalpingogram, day 2 follicle-stimulating hormone &#60; 10 IU/L, mid-luteal progesterone &#62; 30 nmol/L, normal thyroid function, and tested negative for lupus anticoagulant and anticardiolipin IgG and IgM antibodies.
To better understand the immunological basis of RM, it would be most desirable to simultaneously quantify and localize the major immune cell types present in the endometrium at the time of implantation. This paper describes the entire protocol from sample preparation, the multiplex optimization with markers for immune cell subtypes, and the scanning of the slides, followed by data analysis with specific reference to detecting endometrial immune cells. Moreover, this paper describes how to determine the density and clustering of the immune cell types simultaneously in the endometrium.

<table><tbody><tr><td>Amplification Diluent</td><td>Perkin Elmer</td><td>FP1498</td><td>Fluorophore diluent buffer</td></tr><tr><td>Antibody diluent</td><td>Perkin Elmer</td><td>ARD1001EA</td><td>Diluting the antibody</td></tr><tr><td>CD3</td><td>Spring Bioscience</td><td>M3072</td><td>Primary antibody</td></tr><tr><td>CD20</td><td>Biocare Medical</td><td>CM004B</td><td>Primary antibody</td></tr><tr><td>CD56</td><td>Leica</td><td>NCL-CD56-504</td><td>Primary antibody</td></tr><tr><td>CD68</td><td>Spring Bioscience</td><td>M5510</td><td>Primary antibody</td></tr><tr><td>Citrate Buffer Solution, pH 6.0 (10x)</td><td>Abcam</td><td>AB64214</td><td>Antigen retrieval solution</td></tr><tr><td>EMSURE Xylene (isomeric mixture)</td><td>Merck</td><td>108297</td><td>Dewaxing</td></tr><tr><td>Ethanol absolute</td><td>Merck</td><td>107017</td><td>Ethyl alcohol for rehydration</td></tr><tr><td>HistoCore BIOCUT Manual Rotary Leica Microtome</td><td>Leica</td><td>RM2125RTS</td><td>Sectioning of paraffin-embedded tissue</td></tr><tr><td>inForm Advanced Image Analysis Software</td><td>Perkin Elmer</td><td>inForm&lt;sup&gt;&amp;reg;&lt;/sup&gt; Tissue Finder Software 2.2.1 (version 14.0)</td><td>Data Analysis software</td></tr><tr><td>Mantra&lt;sup&gt;&amp;reg;&lt;/sup&gt; Workstation</td><td>Akoya Biosciences</td><td>CLS140089</td><td>Spectral imaging</td></tr><tr><td>Microwave</td><td>Panasonic</td><td>Inverter</td><td>Microwave stripping</td></tr><tr><td>Opal 520</td><td>Perkin Elmer</td><td>FP1487A</td><td>Appropriate tyramide based fluorescent reagent</td></tr><tr><td>Opal 620</td><td>Perkin Elmer</td><td>FP1495A</td><td>Appropriate tyramide based fluorescent reagent</td></tr><tr><td>Opal 650</td><td>Perkin Elmer</td><td>FP1496A</td><td>Appropriate tyramide based fluorescent reagent</td></tr><tr><td>Opal 690</td><td>Perkin Elmer</td><td>FP1497A</td><td>Appropriate tyramide based fluorescent reagent</td></tr><tr><td>Oven</td><td>Memmert</td><td>U10</td><td>Dewaxing</td></tr><tr><td>Peroxidase Blocking Solution</td><td>DAKO</td><td>S2023</td><td>Removal of tissue peroxidase activities</td></tr><tr><td>Poly-L-lysine coated slide</td><td>FISHER SCIENTIFIC</td><td>120-550-15</td><td>Slide for routine histological use</td></tr><tr><td>PolyHRP Broad Spectrum</td><td>Perkin Elmer</td><td>ARH1001EA</td><td>Secondary antibody</td></tr><tr><td>ProLong&amp;trade; Gold Antifade Mountant</td><td>ThemoFisher Scientific</td><td>P36930</td><td>Mounting</td></tr><tr><td>Spatstat</td><td>/</td><td>Version 2.1-0</td><td>Spatial point pattern analysis</td></tr><tr><td>Spectral DAPI</td><td>Perkin Elmer</td><td>FP1490A</td><td>Nucleic acid staining</td></tr><tr><td>Tissue Processor</td><td>Thermo Fischer</td><td>Excelsior ES</td><td>Tissue processing for dehydration and paraffination</td></tr><tr><td>Tris Buffer Saline (TBS), 10x</td><td>Cell Signaling Technology</td><td>12498S</td><td>Washing solution</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P1370-1L</td><td>Nonionic detergent</td></tr></tbody></table>

multiplexed fluorescent immunohistochemical staining of four endometrial immune cell types in recurrent miscarriage

Immunohistochemistry is the most commonly used method for the identification and visualization of tissue antigens in biological research and clinical diagnostics. It can be used to characterize various biological processes or pathologies, such as wound-healing, immune response, tissue rejection, and tissue-biomaterial interactions. However, the visualization and quantification of multiple antigens (especially for immune cells) in a single tissue section using conventional immunohistochemical (IHC) staining remains unsatisfactory. Hence, multiplexed technologies were introduced in recent years to identify multiple biological markers in a single tissue sample or an ensemble of different tissue samples. 
These technologies can be especially useful in differentiating the changes in immune cell-to-cell interactions within the endometrium between fertile women and women with recurrent miscarriages during implantation. This paper describes a detailed protocol for multiplexed fluorescence IHC staining to investigate the density and clustering of four major immune cell types simultaneously in precisely timed endometrial specimens during embryo implantation. The method includes sample preparation, multiplex optimization with markers for immune cell subtypes, and the scanning of the slides, followed by data analysis, with specific reference to detecting endometrial immune cells. 
Using this method, the density and clustering of four major immune cell types in the endometrium can be simultaneously analyzed in a single tissue section. In addition, this paper will discuss the critical factors and troubleshooting to overcome possible fluorophore interference between the fluorescent probes being applied. Importantly, the results from this multiplex staining technique can help provide an in-depth understanding of the immunologic interaction and regulation during embryo implantation.

The overall schematic process of performing a 4-color multiplex assay for the detection of 4 endometrial immune cell types is shown in Figure 1. In brief, the protocol for this multiplex immunofluorescence staining required 8 key steps: 1. Slide preparation, 2. Epitope retrieval, 3. Blocking, 4. Primary antibody application, 5. Secondary antibody application, 6. Signal amplification, 7. Removal of antibody, and 8. Counterstain and mount. Image rendering and analysis were then conducted using...

Watch this Scientific Journal Video about Multiplexed Fluorescent Immunohistochemical Staining of Four Endometrial Immune Cell Types in Recurrent Miscarriage at JoVE.com

Multiplexed Fluorescent Immunohistochemical Staining of Four Endometrial Immune Cell Types in Recurrent Miscarriage

Despite the advancements in multiplex immunohistochemistry and multispectral imaging, characterizing the density and clustering of major immune cells simultaneously in the endometrium remains a challenge. This paper describes a detailed multiplex staining protocol and imaging for the simultaneous localization of four immune cell types in the endometrium.

Immunohistochemistry is the most commonly used method for the identification and visualization of tissue antigens in biological research and clinical ...

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Unit 1

The Endocrine System

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3.3K Views.  The Chinese University of Hong Kong, Prince of Wales Hospital. De huidige technologie maakt het gebruik van antilichamen in dezelfde soort mogelijk voor het detecteren van maximaal zeven markers. De procedure wordt gedemonstreerd door Loucia Chan, een technicus van het laboratorium. Begin met het voorbereiden van de formaline-gefixeerde paraffine ingebedde glazen dia's gemonteerd met het weefselmonster, door de dia's plat in een oven te plaatsen, om te bakken op 60 graden Celsius met het weefsel naar boven gericht.Na een uur ontwas en rehydrateer ...

Video: Multiplexed Fluorescent Immunohistochemical Staining of Four Endometrial Immune Cell Types in Recurrent Miscarriage

Staining Proteins in Gels

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for detecting proteins by four popular methods. Coomassie blue staining is an easy and rapid method. Silver staining, while more time consuming, is considerably more sensitive and can thus be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and is often as sensitive as silver staining. Staining of proteins with SYPRO Orange and SYPRO Ruby are also demonstrated here.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. Staining of proteins with Coomassie Blue, Silver Staining, SYPRO Orange, SYPRO Ruby are demonstrated in this video.

Following separation by electrophoretic methods, proteins in a gel can be detected by several staining methods. This unit describes protocols for ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

In Situ Immunohistochemical Detection of Immune Cell Markers in Cancer Tissues

Multiplex Cyclic Fluorescent Immunohistochemistry

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

Author Spotlight: Efficient Detection of Immune Cell-Infiltration in Cancer Tissues Using Fluorescent Immunohistochemistry

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

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and ...

Cancer Research

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Exploring Uterine NK Cell Heterogeneity in the Endometrium with Multiplex IHC

Measurement of Four Uterine NK Cell Subtypes Using Multiplexed Fluorescent Immunohistochemical Staining in Women with Repeated Implantation Failure

Immunohistochemistry (IHC) plays a crucial role in biological research and clinical diagnosis, serving as the most commonly used method for identifying and visualizing tissue antigens. However, traditional IHC staining methods have limitations in distinguishing various subtypes of immune cells. This challenge has driven scientists to explore new technologies and methodologies for precise identification and differentiation of immune cell subtypes. In recent years, multiplex IHC has emerged as a solution, enabling the simultaneous detection of multiple antigens and their visualization within the same tissue sample. Uterine natural killer (uNK) cells play a pivotal role in early pregnancy processes, including decidualization, remodeling of uterine spiral arteries, and embryo implantation. Different subtypes of uNK cells exhibit different functions, allowing them to coordinate various biological events for successful embryo development and pregnancy. Therefore, in-depth research on uNK cell subtypes is essential for elucidating immune regulation mechanisms during pregnancy. Such studies provide valuable insights and novel approaches for addressing related conditions such as infertility and recurrent reproductive failure. This paper introduces a detailed multiplex IHC staining protocol for studying the density of four subtypes of uNK cells in endometrial specimens during the window of implantation (WOI). The protocol includes sample preparation, optimization of subtype markers, microscopic imaging, and data analyses. This multiplex IHC staining protocol offers high specificity and sensitivity, enabling simultaneous detection of different uNK cell subtypes, thus providing researchers with a powerful tool to explore the intricacies and mechanisms of immune regulation during pregnancy.

Author Spotlight: Multiplex Immunohistochemistry for Understanding Immune Regulation by Uterine NK Cells in Pregnancy

This study presents a pioneering method for quantifying uterine natural killer cell subsets during the window of implantation using advanced multiplexed fluorescent immunohistochemical staining techniques.

Immunohistochemistry (IHC) plays a crucial role in biological research and clinical diagnosis, serving as the most commonly used method for identifying ...

Medicine

Image Analysis of Immunohistochemically Stained Endometrial Tissue

Platform for Quantitative Detection of Endometrial Immune Cells Based on Immunohistochemistry and Digital Image Analysis

To evaluate the endometrial immune microenvironment of patients with recurrent miscarriage (RM), a digital immunohistochemistry image analysis platform was developed and validated to quantitatively analyze endometrial immune cells during the mid-luteal phase. All endometrium samples were collected during the mid-luteal phase of the menstrual cycle. Paraffin-embedded endometrial tissues were sectioned into 4 &#181;m thick slides, and immunohistochemistry (IHC)staining was carried out for detecting endometrial immune cells, including CD56+ uNK cells, Foxp3+ Tregs, CD163+ M2 macrophages, CD1a+ DCs, and CD8+ T cells. The panoramic slides were scanned using a digital slide scanner and a commercial image analysis system was used for quantitative analysis. The percentage of endometrial immune cells was calculated by dividing the number of immune cells in the total endometrial cells. Using the commercial image analysis system, quantitative evaluation of endometrial immune cells, which are difficult or impossible to analyze with conventional image analysis, could be easily, and accurately analyzed. This methodology can be applied to quantitatively characterize the endometrium microenvironment, including interaction between immune cells, and its heterogeneity for different reproductive failure patients. The platform for quantitative evaluation of endometrial immune cells may be of important clinical significance for the diagnosis and treatment of RM patients.

Author Spotlight: Advancing Reproductive Immunology with a Protocol for the Quantitative Evaluation of Endometrial Immune Cells 

Here, a digital immunohistochemistry image analysis platform was developed and validated to quantitatively analyze the endometrial immune cells of patients with recurrent miscarriages in the window of implantation.

To evaluate the endometrial immune microenvironment of patients with recurrent miscarriage (RM), a digital immunohistochemistry image analysis platform ...

Multiplexed Fluorescent Immunohistochemical Staining of Four Endometrial Immune Cell Types in Recurrent Miscarriage

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