Cell Type-Specific Relationships Among Immune Cells in Human Aortic Dissection Tissue.

BACKGROUND: Although recent studies have revealed the importance of inflammation in the pathogenesis of aortic dissection (AD), little is known about the relationships among inflammatory cells in human AD tissue. METHODS AND RESULTS: We assessed the relationships among various immune cell types, including neutrophils, macrophages (M1 and M2), B cells, and helper T cells (Th1, Th2, Th17, Treg and Tfh ) in human AD tissue. AD tissues displayed abundant infiltration of immune cells. Correlation analysis revealed two groups of highly correlated cell types: a group of neutrophils and M1 and M2 macrophages, and another group consisting of B cells and helper T cells. In one particular case of AD, we were able to analyze the correlations between neutrophils and M1 and M2 macrophages in the entry, border, and intact zones of the AD lesions. Neutrophils showed significant correlations with M1 and M2 macrophages in the border zones. The entry and border zones showed M1-dominant polarization, whereas the intact zone showed M2-dominant polarization. CONCLUSIONS: These findings indicate the existence of cell type-specific and site-specific interactions among immune cell types in human AD tissues.

Histological differences related to autophagy in the minor salivary gland between primary and secondary types of Sjögren's syndrome.

Some forms of Sjögren's syndrome (SS) follow a clinical course accompanied by systemic symptoms caused by lymphocyte infiltration and proliferation in the liver, kidneys, and other organs. To better understand the clinical outcomes of SS, here we used minor salivary gland tissues from patients and examine their molecular, biological, and pathological characteristics. A retrospective study was performed, combining clinical data and formalin-fixed paraffin-embedded (FFPE) samples from female patients over 60 years of age who underwent biopsies at Okayama University Hospital. We employed direct digital RNA counting with nCounter® and multiplex immunofluorescence analysis with a PhenoCycler™ on the labial gland biopsies. We compared FFPE samples from SS patients who presented with other connective tissue diseases (secondary SS) with those from stable SS patients with symptoms restricted to the exocrine glands (primary SS). Secondary SS tissues showed enhanced epithelial damage and lymphocytic infiltration accompanied by elevated expression of autophagy marker genes in the immune cells of the labial glands. The close intercellular distance between helper T cells and B cells positive for autophagy-associated molecules suggests accelerated autophagy in these lymphocytes and potential B cell activation by helper T cells. These findings indicate that examination of FFPE samples from labial gland biopsies can be an effective tool for evaluating molecular histological differences between secondary and primary SS through multiplexed analysis of gene expression and tissue imaging.

Toxic effects of dibutyl phthalate on testes of adult zebrafish: evaluation of oxidative stress parameters and histopathology.

Dibutyl phthalate (DBP) is a phthalic compound and is most commonly used as a plasticizer in the polymer industry. It affects the hypothalamus-pituitary-gonadal axis and produces infertility in exposed animals. A total of 366 adult male zebrafish were used to evaluate the toxicological effects of DBP in testes following continuous exposure for 28 days. To evaluate histological changes during phase I of the study, 30 zebrafish were equally divided into five groups viz., control (RO water), vehicle control (0.01% DMSO), T0 (250 µg/L of water), T1 (500 µg/L of water), and T2 group (1000 µg/L of water). The protocol for phase II of the study was decided based on the results of phase I of the study. During phase II, for evaluation of oxidative stress parameters and gene expression profile, a total of 336 fish were equally divided into four groups viz., control, vehicle control, T1 (500 µg/L of water), and T2 (1000 µg/L of water). The activity of SOD, CAT, and TAC was significantly lower in zebrafish from the T2 group; however, a significantly increased level of MDA in the T2 group was recorded as compared to control groups. mRNA expression profile of sod, cat, and nrf2 genes was significantly downregulated in the T2 group as compared to the control group. Histopathology and proliferating cell nuclear antigen immunostaining revealed a reduction in spermatozoa with increased spermatocytes and spermatogonia in testes from T1 and T2 groups. The result indicated that DBP can induce oxidative stress and affect spermatogenesis in zebrafish testes.

Malignant Melanoma With Neural Marker-Positive Distant Organ Cancers.

Malignant melanoma is a melanocyte-derived tumor known for its aggressive clinical behavior. Melanocytes originate from the neural crest, which also gives rise to neural tissues. Malignant melanoma can occasionally exhibit neural differentiation. We report a case of a 70-year-old male with malignant melanoma exhibiting neural marker positivity in the absence of typical melanoma markers. The patient initially presented with a dark nodule on his left heel, which was confirmed as malignant melanoma through cytology. Surgical resection and lymph node dissection were performed, revealing atypical melanocytes. Despite postoperative nivolumab treatment, metastases in the brain and lungs were observed. Histological examination of the brain tumor showed neural differentiation markers (thyroid transcription factor 1 (TTF-1), cytokeratin 7 (CK7), AE1/AE3, and epidermal growth factor receptor (EGFR)) with negative melanoma markers. The patient eventually succumbed to the disease despite multiple treatments. An autopsy revealed multiple organ tumors (brain, duodenum, stomach, liver, and bile duct) negative for melanoma markers but positive for neuroendocrine markers (CD56, synaptophysin, and chromogranin A). This case suggests two possibilities: the coexistence of malignant melanoma with neuroendocrine tumors or a transformation of melanoma into a neuroendocrine phenotype. This case highlights the need for clinicians to consider the potential for melanoma to lose typical markers and transform into neuroendocrine cancer.

Methods and Protocol for Single-Cell Motility Assays under Topological Constraints.

The extracellular environment plays a crucial role in many physiological and pathological processes involving cell motility, such as metastatic invasion in cancer development, by heavily impacting the migration strategies adopted by the cells. The study of how mechanical constraints affect the dynamics of cell migration may be relevant to gain more insight into such processes, and it may prove to be a powerful tool in the hands of biologists. In this chapter, we describe the methods used to investigate the ability of neoplastic cells to migrate through narrowing, rigid microstructures upon chemoattractant stimulation.

Decellularization of Rat Submandibular Gland for Salivary Gland Tissue-Engineering Applications.

OBJECTIVES: This study describes a robust and versatile method for decellularization of rat submandibular glands (SMGs). METHODS: Briefly, rat SMGs were harvested and subjected to perfusion cycles using an anionic detergent. Native and decellularized SMG tissues were subjected to histological analysis using hematoxylin and eosin (H&E) stain and immunohistochemical staining using Hoescht reagent. Further, complementary DNA was synthesized using the native and decellularized SMG tissues and subjected to quantitative reverse transcription polymerase chain reaction (RT-PCR) using rat-specific genes (i.e., α-amylase [Amyl], aquaporin 5 [Aqp5], mucin 19 [Muc19] and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]). The total DNA within native and decellularized SMG tissues were also quantified. RESULTS: H&E staining of SMG tissues revealed preserved ECM content. Decellularized SMG scaffolds lacked cellular material but retained collagen bundles similar to native SMGs. Hoechst reagent immunostaining showed cell nuclei and DNA present in native SMG but not in decellularized SMG scaffolds. Quantitative RT-PCR analysis showed specific amplification products of salivary gland-specific genes (Amyl, Muc19 and Aqp5) and GAPDH in the native SMG tissues. However, no amplification product was observed in the cDNAs from the decellularized SMG scaffolds, confirming the absence of DNA. Quantification of the DNA content showed that the decellularized SMG scaffolds had significantly lower DNA content than native SMG tissue. CONCLUSIONS: Results from this study demonstrated that the decellularization protocol was effective in removing cellular material while preserving the extracellular matrix components and structural integrity of the native SMG tissue.

Identification of cellular and noncellular components of mature intact human peripheral nerve.

BACKGROUND AND AIMS: The goal of this study was to define basic constituents of the adult peripheral nervous system (PNS) using intact human nerve tissues. METHODS: We combined fluorescent and chromogenic immunostaining methods, myelin-selective fluorophores, and routine histological stains to identify common cellular and noncellular elements in aldehyde-fixed nerve tissue sections. We employed Schwann cell (SC)-specific markers, such as S100ß, NGFR, Sox10, and myelin protein zero (MPZ), together with axonal, extracellular matrix (collagen IV, laminin, fibronectin), and fibroblast markers to assess the SC's relationship to myelin sheaths, axons, other cell types, and the acellular environment. RESULTS: Whereas S100ß and Sox10 revealed mature SCs in the absence of other stains, discrimination between myelinating and non-myelinating (Remak) SCs required immunodetection of NGFR along with axonal and/or myelin markers. Surprisingly, our analysis of NGFR+ profiles uncovered the existence of at least 3 different novel populations of NGFR+/S100ß- cells, herein referred to as nonglial cells, residing in the stroma and perivascular areas of all nerve compartments. An important proportion of the nerve's cellular content, including circa 30% of endoneurial cells, consisted of heterogenous S100ß negative cells that were not associated with axons. Useful markers to identify the localization and diversity of nonglial cell types across different compartments were Thy1, CD34, SMA, and Glut1, a perineurial cell marker. INTERPRETATION: Our optimized methods revealed additional detailed information to update our understanding of the complexity and spatial orientation of PNS-resident cell types in humans.

Disomic chromosome 3R(3B) substitution causes a complex of meiotic abnormalities in bread wheat Triticum aestivum L.

Triticum aestivum L. lines introgressed with alien chromosomes create a new genetic background that changes the gene expression of both wheat and donor chromosomes. The genes involved in meiosis regulation are localized on wheat chromosome 3B. The purpose of the present study was to investigate the effect of wheat chromosome 3B substituted with homoeologous rye chromosome 3R on meiosis regulation in disomically substituted wheat line 3R(3B). Employing immunostaining with antibodies against microtubule protein, α-tubulin, and the centromere-specific histone (CENH3), as well as FISH, we analyzed microtubule cytoskeleton dynamics and wheat and rye 3R chromosomes behavior in 3R(3B) (Triticum aestivum L. variety Saratovskaya 29 × Secale cereale L. variety Onokhoiskaya) meiosis. The results revealed a set of abnormalities in the microtubule dynamics and chromosome behavior in both first and second divisions. A feature of metaphase I in 3R(3B) was a decrease in the chiasmata number compared with variety Saratovskaya 29, 34.9 ± 0.62 and 41.92 ± 0.38, respectively. Rye homologs 3R in 13.18 % of meiocytes did not form bivalents. Chromosomes were characterized by varying degrees of compaction; 53.33 ± 14.62 cells lacked a metaphase plate. Disturbances were found in microtubule nucleation at the bivalent kinetochores and in their convergence at the spindle division poles. An important feature of meiosis was the asynchronous chromosome behavior in the second division and dyads at the telophase II in 8-13 % of meiocytes, depending on the anther studied. Considering the 3R(3B) meiotic phenotype, chromosome 3B contains the genes involved in the regulation of meiotic division, and substituting 3B3B chromosomes with rye 3R3R does not compensate for their absence.

Thymosin ß4 and ß10 Expression in Human Organs during Development: A Review.

This review summarizes the results of a series of studies performed by our group with the aim to define the expression levels of thymosin ß4 and thymosin ß10 over time, starting from fetal development to different ages after birth, in different human organs and tissues. The first section describes the proteomics investigations performed on whole saliva from preterm newborns and gingival crevicular fluid, which revealed to us the importance of these acidic peptides and their multiple functions. These findings inspired us to start an in-depth investigation mainly based on immunochemistry to establish the distribution of thymosin ß4 and thymosin ß10 in different organs from adults and fetuses at different ages (after autopsy), and therefore to obtain suggestions on the functions of ß-thymosins in health and disease. The functions of ß-thymosins emerging from these studies, for instance, those performed during carcinogenesis, add significant details that could help to resolve the nowadays so-called "ß-thymosin enigma", i.e., the potential molecular role played by these two pleiotropic peptides during human development.

Detection of ROS in Dormant Breast Cancer Cell.

Reactive oxygen species (ROS) production can occur both as a physiological response and because of oxidative stress. ROS are not only the end product of nonfunctional cell processes but also signaling molecules that can regulate cell and tissue homeostasis. Recently, we have discovered that metastatic breast cancer cells that lay dormant in the lung microenvironment activate mitochondrial ROS production in response to the mechanical properties of the ECM, which triggers an antioxidant response mediated by the NRF2 transcription factor. In turn, this response protects dormant metastatic cells from cisplatin chemotherapy. Many tools have been developed to monitor ROS production in cells in culture, while our ability to detect this in vivo remains limited. Here we describe a detailed protocol for determination of ROS in metastatic cells in the mouse lung tissue by detecting 4-hydroxy-2-noneal (4HNE) adducts formation in fixed tissues.

Automated determination of 8-OHdG in cells and tissue via immunofluorescence using a specially created antibody.

Despite powerful DNA repair systems, oxidative damage/modification to DNA is an inevitable side effect of metabolism, ionizing radiation, lifestyle habits, inflammatory pathologies such as type-2 diabetes or metabolic syndrome, cancer and natural aging. One of the most common oxidative DNA modifications is 8-OHdG (8­hydroxy-2'-deoxyguanosine), which is the most widely used marker in research and clinical diagnostics. 8-OHdG is easily and specifically detectable in various samples such as urine, plasma, cells and tissues via a large variety of methods like ELISA, HPLC, chromatographic methods, and immunochemistry. Formed by oxidation of guanine and being representative for the degree of DNA damage, 8-OHdG can be also used as biomarker for risk assessment of various cancers as well as degenerative diseases. Here, we present a highly specific, self-developed 8-OHdG antibody in successful comparison to a commercially one, tested in cells (FF95, HCT116, and HT22) and intestinal tissue, focusing on automatized evaluation via fluorescence/confocal microscopy.

The Diagnostic Accuracy of Electrical Impedance Spectroscopy-Assisted Colposcopy, HPV mRNA Test, and P16/Ki67 Immunostaining as CIN2+ Predictors in Greek Population.

OBJECTIVE: To evaluate the diagnostic accuracy of Electrical Impedance Spectroscopy (EIS)-assisted colposcopy in detecting CIN2+ Greek women towards standalone colposcopy, HPV mRNA testing, and p16/Ki67 immunostaining. METHODS: We conducted a cross-sectional observational study at the Cervical Pathology Clinic of the 2nd Obstetrics-Gynecology University Department of Hippokration Hospital Thessaloniki involving 316 patients from January 2022 to August 2023. All participants provided liquid-based cervical samples for cytology, HPV mRNA testing, and p16/Ki67 immunostaining. MAIN OUTCOME MEASURES: Subsequently, participants underwent both standalone colposcopy and EIS/ZedScan-assisted colposcopy, followed by cervical punch biopsies. RESULTS: The incorporation of EIS significantly enhanced the sensitivity of colposcopy, increasing it from 54.17% to 100%, equivalent to that of HPV mRNA testing and p16/Ki67 immunostaining, while achieving a high specificity (95.45%). The specificities observed with EIS/ZedScan-assisted and standalone colposcopy were notably superior to those of HPV-related biomarkers (HPV mRNA test and p16/Ki67 immunostaining). When compared to standalone colposcopy, HPV mRNA testing, and p16/Ki67 immunostaining, EIS/ZedScan-assisted colposcopy demonstrated the most favorable combination of Positive and Negative Predictive Values, at 90.57% and 100%, respectively. The inclusion of EIS/ZedScan in colposcopy led to the detection of 44 additional cases of true CIN2+ (100% of the total CIN2+ confirmed histologically) that were missed by standalone colposcopy. This discovery suggests a 45.83% increase in the detection of CIN2+ cases. CONCLUSIONS: The integration of EIS with colposcopy has demonstrated effectiveness in detecting cervical lesions, resulting in a significant detection increase of CIN2+ cases while offering optimal levels of sensitivity, specificity, and predictive values for CIN2+ detection.

Impairment of the GABAergic system in the anterior insular cortex of heroin-addicted males.

Opioid addiction is a global problem, causing the greatest health burden among drug use disorders, with opioid overdose deaths topping the statistics of fatal overdoses. The multifunctional anterior insular cortex (AIC) is involved in inhibitory control, which is severely impaired in opioid addiction. GABAergic interneurons shape the output of the AIC, where abnormalities have been reported in individuals addicted to opioids. In these neurons, glutamate decarboxylase (GAD) with its isoforms GAD 65 and 67 is a key enzyme in the synthesis of GABA, and research data point to a dysregulation of GABAergic activity in the AIC in opioid addiction. Our study, which was performed on paraffin-embedded brains from the Magdeburg Brain Bank, aimed to investigate abnormalities in the GABAergic function of the AIC in opioid addiction by densitometric evaluation of GAD 65/67-immunostained neuropil. The study showed bilaterally increased neuropil density in layers III and V in 13 male heroin-addicted males compared to 12 healthy controls, with significant U-test P values for layer V bilaterally. Analysis of confounding variables showed that age, brain volume and duration of formalin fixation did not confound the results. Our findings suggest a dysregulation of GABAergic activity in the AIC in opioid addiction, which is consistent with experimental data from animal models and human neuroimaging studies.

PIT-EASY survey: validation of the European Pituitary Pathology Group proposal for reporting pituitary neuroendocrine tumors.

The aim of this multicenter prospective survey called PIT-EASY was to assess the relevance of the European Pituitary Pathology Group (EPPG) diagnostic tools for pituitary neuroendocrine tumors (PitNETs) to improve the quality of their histological diagnosis. Each center performed at least 30 histological cases of PitNETs using the EPPG tools and assessed their value using a scorecard with 10 questions. For each center, the histological cases were carried out by pathologists with varying levels of expertise in pituitary pathology defined as junior, intermediate, and expert. Two hundred and ninety histological cases were collected from six French and Italian centers. The three EPPG tools were validated and regarded as helpful for a more accurate and time-efficient diagnosis. The usefulness of level 2 and level 3 of the "EPPG's multi-step approach for immunohistochemistry" including pituitary transcription factors (PIT1, TPIT, and SF1) and chromogranin, SSTRs, and P53 respectively was higher in "other non-functioning" (silent plurihormonal PIT1, silent corticotroph, and null cell): 88% vs 32%, p < 10-6 and 42% vs 14%, p = 0.002, respectively. The diagnostic algorithm proved more useful for junior pathologists (p = 0.0001) and those with intermediate experience. PIT-EASY survey confirmed the importance of a standardized approach to PitNETs for an accurate and reproducible diagnosis and served as validation of the EPPG proposal. The tool appeared to be of practical value to junior participants and staff with intermediate experience for safe routine diagnostic reporting.

Do Fibroblast Growth Factor Receptor (FGFR) 2 and 3 Proteins Play a Role in Prognosis of Invasive Urothelial Bladder Carcinoma?

Background & Objective: Bladder carcinoma ranks second in prevalence among males in Egypt. As a family of tyrosine kinases, fibroblast growth factor receptor (FGFR) dysregulation has been linked to some malignancies in humans. The aim of this study is to analyze the clinicopathological data of patients while investigating FGFR2 and FGFR3 immunohistochemical expression in invasive urothelial bladder carcinoma. Methods: This retrospective cross-sectional study included 60 invasive urothelial carcinoma (UC) cases in the Pathology department, Faculty of Medicine, Menoufia University, from 2009 to 2020. All biopsies were stained for FGFR2 and FGFR3 antibodies. Complete clinical data were available for 44 patients treated and followed in clinical oncology and nuclear medicine departments. Results: Advanced stage and high grade are significantly correlated with FGFR2 positivity (P=0.048 and 0.044, respectively). Cases presented with Perineural invasion showed a higher percentage of FGFR2 (P=0.023). There is a significant indirect linear correlation between FGFR3 expression and lymph node positivity (r= -0.265, P=0.041). Conclusion: A high FGFR2 expression could be associated with poor prognostic parameters, while high FGFR3 expression would be associated with good prognostic parameters. These findings might highlight the importance of FGFR-targeted therapy as a FGFR2 antagonist and FGFR3 agonist for the treatment of urothelial carcinoma patients.

A comparison of fixation and immunofluorescence protocols for successful reproducibility and improved signal in human left ventricle cardiac tissue.

Immunohistochemistry (IHC) and immunofluorescence (IF) are crucial techniques for studying cardiac physiology and disease. The accuracy of these techniques is dependent on various aspects of sample preparation and processing. However, standardised protocols for sample preparation of tissues, particularly for fresh-frozen human left ventricle (LV) tissue, have yet to be established and could potentially lead to differences in staining and interpretation. Thus, this study aimed to optimise the reproducibility and quality of IF staining in fresh-frozen human LV tissue by systematically investigating crucial aspects of the sample preparation process. To achieve this, we subjected fresh-frozen human LV tissue to different fixation protocols, primary antibody incubation temperatures, antibody penetration reagents, and fluorescent probes. We found that neutral buffered formalin fixation reduced image artefacts and improved antibody specificity compared to both methanol and acetone fixation. Additionally, incubating primary antibodies at 37°C for 3 h improved fluorescence intensity compared to the commonly practised 4°C overnight incubation. Furthermore, we found that DeepLabel, an antibody penetration reagent, and smaller probes, such as fragmented antibodies and Affimers, improved the visualisation depth of cardiac structures. DeepLabel also improved antibody penetration in CUBIC cleared thick LV tissue fragments. Thus, our data underscores the importance of standardised protocols in IF staining and provides various means of improving staining quality. In addition to contributing to cardiac research by providing methodologies for IF, the findings and processes presented herein also establish a framework by which staining of other tissues may be optimised.

A Rare Case of Bladder Metastasis From Endometrial Cancer Treated With Robot-Assisted Radical Cystectomy.

Malignant tumors metastasizing to the bladder are uncommon, and bladder metastasis from uterine cancer is particularly rare. Several cases of bladder metastasis from malignant melanoma, gastric cancer, breast cancer, and renal cancer have been documented. However, to our knowledge, only four cases of bladder metastasis from endometrial cancer had been reported up until 2024. Here, we present a case of bladder metastasis of endometrial cancer following modified radical hysterectomy, which was successfully treated through multidisciplinary intervention.

Genome-wide sequence divergence of satellite DNA could underlie meiotic failure in male hybrids of bighead catfish and North African catfish (Clarias, Clariidae).

Hybrid sterility, a hallmark of postzygotic isolation, arises from parental genome divergence disrupting meiosis. While chromosomal incompatibility is often implicated, the underlying mechanisms remain unclear. This study investigated meiotic behavior and genome-wide divergence in bighead catfish (C. macrocephalus), North African catfish (C. gariepinus), and their sterile male hybrids (important in aquaculture). Repetitive DNA analysis using bioinformatics and cytogenetics revealed significant divergence in satellite DNA (satDNA) families between parental species. Notably, one hybrid exhibited successful meiosis and spermatozoa production, suggesting potential variation in sterility expression. Our findings suggest that genome-wide satDNA divergence, rather than chromosome number differences, likely contributes to meiotic failure and male sterility in these catfish hybrids.

Moxidectin versus Ivermectin in the prevention and treatment of acute and chronic experimental trichinellosis.

The limited activity of the traditional medications against T. spiralis encysted larvae handicaps complete cure of trichinellosis till now due to decreased permeability and absorption through tissues. MOX is listed worldwide for prevention and treatment of several internal and external nematodes. Consequently, the aim of this work was to investigate the effect of moxidectin versus ivermectin on experimental acute and chronic trichinellosis and to illuminate the potential mechanisms of their effects. 105 Mice were divided into four groups; Group I: Uninfected healthy control; Group II: Infected untreated control; Group III: Infected and treated with IVM and Group IV: Infected and treated with MOX. The groups (II, III and IV) were later subdivided equally into three subgroups (a, b, and c) according to the stage of treatment. Parasitological counting of adults and larvae besides immune-histopathological examination of intestines and muscles were done. Results exhibited that both IVM and MOX succeeded in reducing adults and larvae counts with higher potential of MOX in both intestinal and muscle phase. The preeminence of MOX was indicated by decreased inflammation, a significant reduction in the microvascular density (CD31 immunostaining) as well as a reduction in the percentage of fibroblast activation protein (FAP) immunostaining in muscle tissues. Accordingly, the current work recommends moxidectin as an innovative treatment for trichinellosis.

Analysis of Surface Expression of NMDAR Subunits in Primary Hippocampal Neurons.

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.