The present study was supported by the Health and Medical Research Fund (10210956).

The study was approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee (CREC ref no.: 2022.581). Women with RIF were recruited from the Assisted Reproductive Technology Center, Prince of Wales Hospital, Chinese University of Hong Kong. RIF was defined as the failure to achieve a clinical pregnancy after the transfer of at least 4 good-quality embryos in a minimum of 3 fresh or frozen cycles in a woman under the age of 40 years10. Informed consent was obtained from the participants before collecting the endometrial biopsies.
1. Acquisition and processing of endometrial samples
<ol>
	<li>Timing of endometrial specimen collection
	<ol>
		<li>Collect endometrial samples following a strict protocol to ensure consistency among the study participants. For patients with natural cycles, conduct urinalysis from the 9th day of the menstrual cycle and perform endometrial biopsy on the 7th day after the LH surge (LH+7)11 . For patients undergoing hormone replacement therapy (HRT) cycles, administer 6 mg of estradiol valerate orally daily starting on day 2 of the menstrual cycle.</li>
		<li>Assess endometrial thickness by ultrasonography on day 13 of the menstrual cycle. When the endometrial thickness reaches or exceeds 8 mm, administer progesterone transvaginally and conduct an endometrial biopsy 5 days after progesterone administration12.</li>
	</ol>
	</li>
	<li>Endometrial sample collection and fixation: introduce a pipette sampler into the uterine cavity, advancing it to the fundal region; apply suction by pulling down the internal plunger, simultaneously scraping the endometrial surface through rotational and vertical movements within the uterine cavity. Rinse the collected specimens with normal saline to eliminate cervical and uterine secretions. Measure samples and assess the size. For 3 mm x 15-25 mm samples, immediately immerse them in 10 mL of 10% neutral buffered formalin and fix them at room temperature for 24-48 h.</li>
	<li>Dehydration: Upon completion of fixation, subject samples to a dehydration sequence designed to replace water with ethanol. To do this, immerse tissues in 70%, 80%, 90%, and 100% ethanol baths to ensure complete dehydration. Following the final ethanol bath, clear the samples in xylene to remove any residual alcohol and prepare them for paraffin infiltration.</li>
	<li>Embedding: After placing the tissue flat on the bottom of the mold, melt paraffin and carefully add it to the samples to ensure that the endometrial tissue is completely encapsulated in the paraffin matrix. After filling the molds with paraffin wax, cool them on a freezer table to promote solidification of the paraffin wax. Once the paraffin blocks harden, store at room temperature until further processing is required. 
	NOTE: The molds used were specifically chosen to accommodate the size of the endometrial samples obtained to allow the endometrial tissue to lay flat on the bottom without folding, thus ensuring optimal orientation for subsequent sectioning and without creating unnecessary excess.</li>
	<li>Sectioning: To prepare for sectioning, refrigerate the paraffin blocks at 4 &#176;C for 2 h. Slice the samples into 4 &#181;m sections using a microtome, followed by flattening the slices on the surface of distilled water warmed to 42 &#176;C and mounting them onto adhesive glass slides. Place the slices on a slide dryer overnight and store at room temperature prior use.</li>
</ol>
2. Optimization of multiplex immunohistochemistry conditions
<ol>
	<li>IHC validation: For the validation of IHC staining parameters, adopt a systematic approach to fine-tune conditions for antigen retrieval, antibody concentration, and incubation times.
	<ol>
		<li>Within the manufacturer&#39;s recommended dilution ranges, test 2-3 concentrations for each antibody. Optimize antigen retrieval by comparing buffers at pH 6 and pH 9, and evaluate incubation protocols at both room temperature for 1.5 h and overnight at 4 &#176;C.</li>
		<li>Assess each condition meticulously, focusing on staining intensity, specificity, and background noise. Select the optimal combination yielding the clearest, most specific staining with minimal non-specific binding. This rigorous validation ensured the highest accuracy and reproducibility in the IHC protocol.</li>
	</ol>
	</li>
	<li>Tyramide Signal Amplification (TSA) monoplex staining: After determining the optimal staining parameters for individual antibodies, optimize the pairing of antibodies with different TSA dyes. Pair the marker with higher expression with a dye with a lower fluorescence intensity level, or vice versa. During this phase, make adjustments to antibody concentration and incubation time according to the observed fluorescence signal strength. If significant background or non-specific staining is observed, change the paired dye13.</li>
	<li>TSA multiplex staining: For the multiplex immunohistochemistry (m-IHC) protocol, the sequential order of staining has been carefully defined according to established principles. To ensure the multiplexing process reflects the sensitivity and specificity of single-label staining, prioritized antibodies with lower expression levels at the beginning of the staining sequence. Place antibodies that require base repair for antigen retrieval at the end of the staining sequence, as base repair is robust compared to acid-based methods, which can, in some cases, amplify non-specific staining artifacts.</li>
	<li>Using the findings from steps 2.1 and 2.2, decide on the antibodies to be used and at what stage, based on their antigen retrieval requirements and expression densities. For example, the CXCR4 antibody, which requires a base repair for antigen retrieval, has been placed in the final stages of staining. Conversely, the CD49a antibody, which is characterized by a lower density of expression, has been given the first position in the staining sequence. Maximize the chances of successful detection and minimize any masking effects that might occur in the later stages of the multiplexing procedure.</li>
	<li>For the remaining antibodies, test different combinations during the optimization phase to determine the most effective sequence that balances both sensitivity and specificity. The results of these comparative analyses will guide the final composition of the multiplex panel, as detailed in Table 1, ensuring that the selected panel provides optimal performance while minimizing background.</li>
</ol>
3. m-IHC process
<ol>
	<li>Dewaxing, hydration, and fixation: Bake the tissue sections at 60 &#176;C for 2 h, followed by sequential immersion in xylene I (10 min), xylene II (10 min), 100% ethanol (5 min), 95% ethanol (5 min), 80% ethanol (5 min), 75% ethanol (5 min), ddH2O (2 min for 3x), 10% neutral buffered formalin (at least 20 min), and ddH2O (2 min for 3x).</li>
	<li>Antigen retrieval: Add 200 mL of the respective AR6 or AR9 buffer, as listed in Table 1 for each antibody, to a heat-resistant antigen retrieval cassette. Place the sections in the appropriate antigen retrieval buffer and heat in a microwave on high power for about 2 min until boiling. Subsequently, cover the cassette loosely, heat on low power for 15 min, and cool to room temperature. Rinse the sections with ddH2O and PBST.</li>
	<li>Drawing hydrophobic barrier: After removing the excess solution with absorbent paper, draw a complete circle around the tissue using a hydrophobic barrier pen.</li>
	<li>Inactivation of endogenous enzyme activity: Treat sections with 3% hydrogen peroxide and incubate for 8 min at room temperature. Wash with PBST 3x for 2 min each.</li>
	<li>Blocking of non-specific sites: After drying with absorbent paper, apply a blocking solution (1x Antibody Diluent/Block) and incubate for 10 min at room temperature.</li>
	<li>Incubation with primary antibody: After removal of the blocking solution, apply 150 &#181;L of the first primary antibody, CD49a, to each slide at a dilution of 1:1000 in 1x Antibody Diluent. Incubate as previously optimized in section 2. After incubation, wash the slides with PBST 3x for 2 min each. For the remaining antibodies, see Table 1 for specific dilution ratios.</li>
	<li>Incubation with secondary antibody: Before applying the secondary antibody, use absorbent paper to remove excess moisture to prevent dilution of the reagent. Add 4-5 drops (approximately 150 &#181;L) of the 1x Anti-Ms + Rb HRP secondary antibody onto each slide, followed by a 10 min incubation at room temperature. Perform three washes with PBST for 2 min each post-incubation. 
	NOTE: Please note that the 1x Anti-Ms + Rb HRP secondary antibody is specific for rabbit and mouse primary antibodies.</li>
	<li>Signal amplification with TSA dye: After blotting excess moisture, dilute the TSA dye 1:100 in the amplification diluent. Add 150 &#181;L of this diluted TSA dye solution to each slide. Incubate for 10 min at room temperature. Wash with PBST 3x for 2 min each.</li>
	<li>Repeat steps 3.2 and 3.5-3.8 for the TSA procedure using the corresponding antibody and dye.</li>
	<li>Staining nuclei: After staining the last antibody, eliminate unbound fluorophores by microwaving sections in 200 mL of AR6 buffer within a heat-resistant cassette, boiling for 2 min, then low-power heating for 15 min. Cool samples to room temperature and rinse with ddH2O and PBST. Add 150 &#181;L of DAPI working solution diluted in PBST (1:10) dropwise, incubate for 10 min at room temperature, and rinse 2x with ddH2O.</li>
	<li>Mounting: Once sections are entirely air-dried in a dark area for approximately 30 min, apply a single drop (around 30 &#181;L) of the anti-quenching fluorescent mountant, and place coverslips. 
	NOTE: Protect all operations from light after the first application of TSA dye. Prompt observation and image capture were essential post-staining completion to prevent fluorescence quenching.</li>
</ol>
4. Image acquisition and analysis
<ol>
	<li>Exposure time: Capture images using an imager that inherits excitation and emission wavelengths corresponding to the fluorophores. When setting exposure times, check slides with a wide range of marker expressions to ensure accuracy. For each marker/filter combination, manually focus on different areas of the slide. Use the Auto Exposure feature to set the exposure time.</li>
	<li>Select filters according to the TSA dyes, and examine multiple slides to determine optimal exposure times per channel. Apply these settings consistently to all subsequent acquisitions to maintain comparability throughout imaging.</li>
	<li>Selection of capture areas: To ensure unbiased sampling across tissue sections and consistent inclusion of the luminal epithelium in each analyzed field, randomly select the initial area for image acquisition, with the critical criterion being the presence of the luminal epithelial border.</li>
	<li>Following the capture of this initial field, systemically select subsequent areas by moving one field to the left or right of the starting point (omitting one field between each image captured) while maintaining the luminal epithelial border within the frame. Repeat this process to capture at least five different areas, each containing at least a portion of the luminal epithelium, to maintain a consistent depth of analysis throughout14. This approach aimed to include a minimum of approximately 10,000 stromal cells across all captured fields to ensure comprehensive and representative data collection.</li>
	<li>Image analysis
	<ol>
		<li>Image preparation: To import and analyze multispectral images, first navigate to File &#62; Open Image in the software interface to load the files and the Autofluorescence (AF) slide. After uploading the images, proceed to unmix the fluorophores by clicking the Select Fluors button and selecting the appropriate spectral library. Once all fluorophores are selected, click OK to confirm the selection. To isolate the AF spectrum, use the AF eyedropper tool to sample a representative area of tissue from the AF image, taking care to exclude any non-tissue pixels. After sampling, click Prepare Image or Prepare All in the lower left corner of the interface15.</li>
		<li>Tissue segmentation: Initiate the segmentation process by manually delineating a baseline of 3-5 regions per tissue type within the image, including epithelial areas containing epithelial cells, stromal areas containing stromal cells, and blank areas without cellular content. If the initial segmentation results are not satisfactory, manually annotate additional regions to improve the training dataset, thereby increasing the accuracy and reliability of subsequent analyses. This dynamic adjustment of regions and parameters ensures that the software can automatically identify similar structures in the current and other imported images with increased accuracy. 
		NOTE: The initial annotation serves as a training dataset for the software to learn the characteristics of each tissue component. The software&#39;s segmentation capabilities are then iteratively refined based on the complexity and variability of the tissue.</li>
		<li>Cell Segmentation: Click Segment Cells to start cell segmentation. In the Cell Segmentation Setting, select Nuclei and Membrane. Choose DAPI to identify cell nuclei and adjust the intensity setting to detect all cell nuclei without background noise. Select CD16, CD49a, and CD56 signals to find the membrane and use this signal to assist in nuclear splitting. Use the nuclear component splitting feature to distinguish closely located nuclei. 
		NOTE: DAPI highlights the inside of cells. The nuclear component splitting feature helps to distinguish cell nuclei when they are close together or look merged. This step is important for getting reliable cell counts and maintaining the integrity of the analysis.</li>
		<li>Cell phenotyping: Adopt four different cell surface markers including CD56, CD49a, CXCR4 and CD16 to differentiate different subtypes of NK cells. In the Phenotyping Scheme, label these four markers as the phenotypes to proceed. In the Associate View section, assign specific colors to each of the phenotypes for identification. Using the Add button, categorize cells as either positively or negatively expressing the markers; for example, in the CD56 phenotype, cells can be labeled as CD56+ or CD56-. Manually label at least five cells for each phenotype during the training process. Perform additional manual labeling if the initial training does not yield effective results.</li>
		<li>After the cell phenotyping configuration is set, each cell in the image will indicate whether the four surface markers are positively or negatively expressed. Export the resulting data to a spreadsheet for further analysis.</li>
		<li>Data processing: Import the data obtained from all images into a spreadsheet for processing. Calculate percentages of all uNK cell subtypes relative to the total number of cells in the image. The final cell count represents the average count across multiple images, providing a comprehensive overview of the population of various uNK cell subtypes within the sample.</li>
	</ol>
	</li>
</ol>

Exploring Uterine NK Cell Heterogeneity in the Endometrium with Multiplex IHC

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

Embryo implantation involves a complex interaction between the embryo and the endometrium. The immunological status of endometrial homeostasis plays a pivotal role in determining endometrial receptivity. During WOI, the predominant leukocyte population in the endometrium is NK cells. Approximately 90% of uNK cells exhibit high CD56 expression but lack CD16. However, a minor subset of uNK cells resembles peripheral blood NK cells, displaying low CD56 expression but positive CD16 expression18. These...

The first documented live birth after in vitro fertilization-embryo transfer (IVF-ET) was reported in 1978. Over the past 40 years, there has been a high demand for the assistance of IVF-ET among infertile couples1. In 2021, 238,126 patients initiated a total of 413,776 IVF cycles in the United States. This marks a 25% increase in cycles from 2 years prior and a 135% increase from 20122. This surge is mainly attributed to the rising prevalence of infertility and delayed planning for pregnancy. Advancements in embryo culture techniques and superovulation protocols have led to an increased live birth rate per ET cycle, reaching 30%-50% in women less than 40 years old and less than 30% in women older than 40 years2. However, despite these advancements, over half of transferred embryos still fail to implant. Repeated implantation failure (RIF), typically defined as failure after three or more consecutive attempts of transferring high-quality embryos, affects 15% of women who undergo IVF-ET3. Couples with RIF are extremely vulnerable and more prone to undergo expensive and unnecessary procedures that can expose them to undue risks4. Therefore, understanding the causes of RIF and improving embryo implantation is crucial to enhance the success of IVF-ET, particularly for women with RIF. The preparation of endometrium is critical for successful embryo implantation. This process is characterized by a significant accumulation of uterine natural killer (uNK) cells, which transition from constituting 30% of total lymphocytes in the endometrium during mid-secretory phase to 70%-80% in the decidua during early pregnancy5. Notably, uNK cells differ from peripheral NK cells, which are cytotoxic lymphocytes critical to the innate immune system for causing the death of the infected cells through lysis or apoptosis. While the exact functions of uNK cells are not yet fully understood, several lines of evidence suggest that they are involved in angiogenesis remodeling, trophoblast invasion, and fetal development6. The association between the percentage of uNK cells over stromal cells and RIF has garnered extensive attraction over the past 20 years. A recent meta-analysis, which included 8 studies involving 604 women, demonstrated that the density of CD56+uNK cells during the mid-luteal phase is significantly increased in women with RIF compared to fertile controls7. However, it is important to note that the characteristics of uNK cells during the mid-luteal phase differ significantly from those of decidual NK (dNK) cells. Although uNK cells may further differentiate into various subsets of dNK cells post-pregnancy, measuring uNK cells alone does not accurately represent dNK cells8. uNK cells undergo dynamic differentiation and play different roles in the menstrual cycle and decidualization processes, making them more complex than can be identified by CD56 alone. Multiple markers are required to achieve a comprehensive understanding of uNK cell behavior during endometrial preparation. Our recent study employed single-cell RNA sequencing to identify the diversity of uNK cells throughout menstrual cycles. The results, validated using flow cytometry, have shown the presence of four distinct subtypes of uNK cells, each exhibiting dynamic changes during the menstrual cycle9. Gene enrichment analysis and gene ontology functional enrichment indicate these uNK subsets fulfill different functions at various stages of menstruation. Nevertheless, flow cytometry is not universally accessible in clinical laboratories, and the immediate processing of fresh endometrial tissue for enzyme digestion renders it impossible to repeat experimental steps upon errors.
The aim of this study was, therefore, to investigate the measurement of these four subpopulations of NK cells using a multiplex staining assay, which provides a more practical diagnostic approach. In multiple staining, different specific antibodies against each target are linked to different fluorophore labels that emit different wavelengths of light when excited by a specific wavelength of light. Compared to the traditional IHC staining method, this method can quantitatively compare the relative abundance and distribution of multiple targets in a sample and provide information on the interaction and co-localization of different targets, enabling us to identify different subtypes of uNK cells. This approach will not only deepen our understanding of the relationship between uNK cells and RIF but will also provide insights for investigating other immune cell subpopulations in endometrial-related diseases.

<table><tbody><tr><td>CD49a</td><td>Novus Biologicals</td><td>NBP2-76478</td><td>Primary antibodies</td></tr><tr><td>CD56</td><td>Leica</td><td>NCL-L-CD56-504</td><td>Primary antibodies</td></tr><tr><td>CD16</td><td>abcam</td><td>ab183354</td><td>Primary antibodies</td></tr><tr><td>CXCR4</td><td>R&amp;amp;D</td><td>MAB172</td><td>Primary antibodies</td></tr><tr><td>Amplification diluent</td><td>Akoya Biosciences</td><td>FP1498 series</td><td>Fluorophore dilution buffer</td></tr><tr><td>Antibody diluents</td><td>Akoya Biosciences</td><td>ARD1001EA</td><td>Dilute the antibody</td></tr><tr><td>Citrate buffered solution, pH 6.0 /9.0(10x)</td><td>Akoya Biosciences</td><td>A6001/A9001</td><td>Antigen retrieval solution</td></tr><tr><td>inForm advanced image analysis software</td><td>Akoya Biosciences</td><td>inForm Tissue Finder Software 2.2.6</td><td>Data analysis software</td></tr><tr><td>Mantra Workstations</td><td>Akoya Biosciences</td><td>CLS140089</td><td>Spectral imaging</td></tr><tr><td>microwave</td><td>Akoya Biosciences</td><td>inverter</td><td>Microwave stripping</td></tr><tr><td>TSA 520</td><td>Akoya Biosciences</td><td>FP1487001K</td><td>Suitable tyramide-based fluorescent reagents</td></tr><tr><td>TSA 620</td><td>Akoya Biosciences</td><td>FP1495001K</td><td>Suitable tyramide-based fluorescent reagents</td></tr><tr><td>TSA 650</td><td>Akoya Biosciences</td><td>FP1496001K</td><td>Suitable tyramide-based fluorescent reagents</td></tr><tr><td>TSA 570</td><td>Akoya Biosciences</td><td>FP1488001K</td><td>Suitable tyramide-based fluorescent reagents</td></tr><tr><td>Poly-L-lysine coated slides</td><td>Fisher Technologies</td><td>120-550-15</td><td>Slides for routine histological use</td></tr><tr><td>PolyHRP Broad Spectrum</td><td>Perkin Elmer</td><td>ARH1001EA</td><td>Secondary antibodies</td></tr><tr><td>Invitrogen&amp;trade;&amp;nbsp;Fluoromount-G&amp;trade; Mounting Medium</td><td>ThemoFisher Science</td><td>495802</td><td>Installation</td></tr><tr><td>Spectral DAPI</td><td>Akoya Biosciences</td><td>FP1490A</td><td>Nucleic acid staining</td></tr></tbody></table>

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.

To maintain consistency in the timing of endometrial sample collection for women undergoing the natural cycle, a urine test was performed to precisely detect their luteinizing hormone (LH) surge, with endometrial biopsies conducted 7 days after the LH surge. For women undergoing HRT cycles, samples were scheduled precisely 5 days after progesterone supplementation commenced. To quantify different subtypes of NK cells in the endometrium, m-IHC staining was employed. A schematic outlining of the experimental procedure is d...

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.

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 ...

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Medicine

1.1K Views.  The Chinese University of Hong Kong. Baarmoeder NK-cellen hopen zich dramatisch op tijdens de mid-luteale fase, voorbereid op embryo-implantatie. Deze cellen zijn met name heterogeen. Het nauwkeurig identificeren van hun fenotypes is cruciaal voor het begrijpen van hun rol in de ontvankelijkheid van het endometrium.Onze studie heeft tot doel vier subtypes van NK-cellen in de baarmoeder te detecteren met behulp van multiplex immunohistochemische kleuring. Immunohistochemische kleuring is o...

Video: Auteur in de schijnwerpers: multiplex immunohistochemie voor het begrijpen van immuunregulatie door baarmoeder NK-cellen tijdens de zwangerschap

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8.
Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12.
In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor&#x2019;s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

B7-H1/PD-L1, a member  of the B7 family of immune-regulatory cell-surface proteins, plays an important  role in the negative regulation of cell-mediated ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated measurements over weeks or months of accessory respiratory muscle activity while simultaneously measuring ventilation in a non-anesthetized, freely behaving mouse. The technique includes the surgical implantation of a radio transmitter and the insertion of electrode leads into the scalene and trapezius muscles to measure the electromyogram activity of these inspiratory muscles. Ventilation is measured by whole-body plethysmography, and animal movement is assessed by video and is synchronized with electromyogram activity. Measurements of muscle activity and ventilation in a mouse model of amyotrophic lateral sclerosis are presented to show how this tool can be used to investigate how respiratory muscle activity changes over time and to assess the impact of muscle activity on ventilation. The described methods can easily be adapted to measure the activity of other muscles or to assess accessory respiratory muscle activity in additional mouse models of disease or injury.

This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...

Isolation of Uterine Innate Lymphoid Cells for Analysis by Flow Cytometry

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow cytometry. The protocol describes how to set up time mating to obtain multiple synchronous dams, the mechanical and enzymatic digestion of the pregnant uterus, the staining of single-cell suspensions, and a FACS strategy to phenotype and discriminate g1 uILCs. Although this method inevitably loses the spatial information of cellular distribution within the tissue, the protocol has been successfully applied to determine uILC heterogeneity, their response to maternal and foetal factors affecting pregnancy, their gene expression profile, and their functions.

This is a method to isolate uterine lymphoid cells from both pregnant and non-pregnant mice. This method can be used for multiple downstream applications such as FACS phenotyping, cell sorting, functional assays, RNA-seq, and proteomics. The protocol here demonstrates how to phenotype group 1 uterine innate lymphoid cells by flow cytometry.

Described here is a simple method to isolate and phenotype mouse group 1 uterine innate lymphoid cells (g1 uILCs) from individual pregnant uterus by flow ...

Immunology and Infection

Implantation of an Isoproterenol Mini-Pump to Induce Heart Failure in Mice

Isoproterenol (ISO), is a non-selective beta-adrenergic agonist, that is used widely to induce cardiac injury in mice. While the acute model mimics stress-induced cardiomyopathy, the chronic model, administered through an osmotic pump, mimics advanced heart failure in humans. The purpose of the described protocol is to create the chronic ISO-induced heart failure model in mice using an implanted mini-pump. This protocol has been used to induce heart failure in 100+ strains of inbred mice. Techniques on surgical pump implantation are described in detail and may be relevant to anyone interested in creating a heart failure model in mice. In addition, the weekly cardiac remodeling changes based on echocardiographic parameters for each strain and expected time to model development are presented. In summary, the method is simple and reproducible. Continuous ISO administered via the implanted mini-pump over 3 to 4 weeks is sufficient to induce cardiac remodeling. Finally, the success for ISO model creation may be assessed in vivo by serial echocardiography demonstrating hypertrophy, ventricular dilation, and dysfunction.

The chronic administration of isoproterenol via an implanted osmotic pump has been used widely to mimic advanced heart failure in mice. Here, we describe detailed methods in surgical mini-pump implantation for the continuous isoproterenol administration over 3 weeks, as well as, echocardiographic assessment for the successful model creation.

Isoproterenol (ISO), is a non-selective beta-adrenergic agonist, that is used widely to induce cardiac injury in mice. While the acute model mimics ...

Repeated Orotracheal Intubation in Mice

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ventral neck for direct confirmation of cannula placement in the trachea. The relative difficulty or the tissue trauma induced to the subject by such procedures can be an impediment to an investigator&#8217;s ability to perform longitudinal studies. This article illustrates a technique in which physical manipulation of the mouse following the use of a depilatory to remove hair from the ventral neck permits transcutaneous visualization of the trachea for orotracheal intubation regardless of degree of skin pigmentation. This method is innocuous to the subject and easily achieved with a limited understanding of murine anatomy. This refined approach facilitates repeated intubation, which may be necessary for monitoring progression of disease or instillation of treatments. Using this method may result in a reduction of the number of animals and technical skill required to measure lung function in mouse models of respiratory disease.

The goal of this article is to describe a refined method of intubation of the laboratory mouse. The method is noninvasive and, therefore, ideal for studies that require serial monitoring of respiratory function and/or instillation of treatments into the lung.

The literature describes several methods for mouse intubation that either require visualization of the glottis through the oral cavity or incision in the ...

Operating Transverse Aortic Constriction with Absorbable Suture to Obtain Transient Myocardial Hypertrophy

Based on twice transverse aortic constrictions (TACs) in mice, it is proved that myocardial hypertrophic preconditioning (MHP) could attenuate cardiomyocyte hypertrophy and slow down progression to heart failure. For novices, however, the MHP model is usually quite difficult to establish because of the technical obstacles in ventilator operation, opening the chest repeatedly, and bleeding caused by debanding. To facilitate this model, to increase the surgical success rate and to reduce the incidence of bleeding, we switched to absorbable sutures for the first TAC combing with a ventilator-free technique. Using a 2-week absorbable suture, we demonstrated that this procedure could cause significant myocardial hypertrophy in 2 weeks; and 4 weeks after surgery, myocardial hypertrophy was almost completely regressed to the baseline. Using this protocol, the operators could master the MHP model easily with a lower operation mortality.

This protocol presents an improved method to obtain transient myocardial hypertrophy with absorbable suture, simulating left ventricular hypertrophy decrease after removing pressure overload. It could be valuable for the studies on myocardial hypertrophic preconditioning.

Based on twice transverse aortic constrictions (TACs) in mice, it is proved that myocardial hypertrophic preconditioning (MHP) could attenuate ...

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.

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 ...

Biology

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 ...

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

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