3D exploration of gene expression in chicken embryos through combined RNA fluorescence in situ hybridization, immunofluorescence, and clearing.

BACKGROUND: Fine characterization of gene expression patterns is crucial to understand many aspects of embryonic development. The chicken embryo is a well-established and valuable animal model for developmental biology. The period spanning from the third to sixth embryonic days (E3 to E6) is critical for many organ developments. Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA-FISH) enables multiplex RNA detection in thick samples including embryos of various animal models. However, its use is limited by tissue opacity. RESULTS: We optimized HCR RNA-FISH protocol to efficiently label RNAs in whole mount chicken embryos from E3.5 to E5.5 and adapted it to ethyl cinnamate (ECi) tissue clearing. We show that light sheet imaging of HCR RNA-FISH after ECi clearing allows RNA expression analysis within embryonic tissues with good sensitivity and spatial resolution. Finally, whole mount immunofluorescence can be performed after HCR RNA-FISH enabling as exemplified to assay complex spatial relationships between axons and their environment or to monitor GFP electroporated neurons. CONCLUSIONS: We could extend the use of HCR RNA-FISH to older chick embryos by optimizing HCR RNA-FISH and combining it with tissue clearing and 3D imaging. The integration of immunostaining makes possible to combine gene expression with classical cell markers, to correlate expressions with morphological differentiation and to depict gene expressions in gain or loss of function contexts. Altogether, this combined procedure further extends the potential of HCR RNA-FISH technique for chicken embryology.

Precise immunofluorescence canceling for highly multiplexed imaging to capture specific cell states.

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and intercellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we develop Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

Single-Cell Single-Molecule RNA-FISH Combined with Immunofluorescence and High-Speed and High-Resolution Scanning Analysis to Visualize the Reactivation of Latent HIV-1.

Latent HIV-1 reservoirs are a major obstacle to the eradication of HIV-1. Several cure strategies have been proposed to eliminate latent reservoirs. One of the key strategies involves the reactivation of latent HIV-1 from cells using latency-reversing agents. However, currently it is unclear whether any of the latency-reversing agents are able to completely reactivate HIV-1 provirus transcription in all latent cells. An understanding of the reactivation of HIV-1 provirus at single-cell single-molecule level is necessary to fully comprehend the reactivation of HIV-1 in the reservoirs. Furthermore, since reactivable viruses in the pool of latent reservoirs are rare, combining single-cell imaging techniques with the ability to visualize a large number of reactivated single cells that express both viral RNA and proteins in a pool of uninfected and non-reactivated cells will provide unprecedented information about cell-to-cell variability in reactivation. Here, we describe the single-cell single-molecule RNA-FISH (smRNA-FISH) method to visualize HIV-1 gag RNA combined with the immunofluorescence (IF) method to detect Gag protein to characterize the reactivated cells. This method allows the visualization of subcellular localization of RNA and proteins before and after reactivation and facilitates absolute quantitation of the number of transcripts per cell using FISH-quant. In addition, we describe a high-speed and high-resolution scanning (HSHRS) fluorescence microscopy imaging method to visualize rare and reactivated cells in a pool of non-reactivated cells with high efficiency.

Novel approach to soft tissue regeneration: in vitro study of compound hyaluronic acid and horizontal platelet-rich fibrin combination.

OBJECTIVE: This study aims to develop a compound biomaterial to achieve effective soft tissue regeneration. METHODOLOGY: Compound hyaluronic acid (CHA) and liquid horizontal-platelet-rich fibrin (H-PRF) were mixed at a ratio of 1:1 to form a CHA-PRF gel. Human gingival fibroblasts (HGFs) were used in this study. The effect of CHA, H-PRF, and the CHA-PRF gel on cell viability was evaluated by CCK-8 assays. Then, the effect of CHA, H-PRF, and the CHA-PRF gel on collagen formation and deposition was evaluated by qRT‒PCR and immunofluorescence analysis. Finally, qRT‒PCR, immunofluorescence analysis, Transwell assays, and scratch wound-healing assays were performed to determine how CHA, H-PRF, and the CHA-PRF gel affect the migration of HGFs. RESULTS: The combination of CHA and H-PRF shortened the coagulation time of liquid H-PRF. Compared to the pure CHA and H-PRF group, the CHA-PRF group exhibited the highest cell proliferation at all time points, as shown by the CCK-8 assay. Col1a and FAK were expressed at the highest levels in the CHA-PRF group, as shown by qRT‒PCR. CHA and PRF could stimulate collagen formation and HGF migration, as observed by fluorescence microscopy analysis of COL1 and F-actin and Transwell and scratch healing assays. CONCLUSION: The CHA-PRF group exhibited greater potential to promote soft tissue regeneration by inducing cell proliferation, collagen synthesis, and migration in HGFs than the pure CHA or H-PRF group. CHA-PRF can serve as a great candidate for use alone or in combination with autografts in periodontal or peri-implant soft tissue regeneration.

Development and evaluation of time-resolved rapid immunofluorescence test for detection of TSOL18 specific antibody in porcine cysticercosis infections.

BACKGROUND: Porcine cysticercosis, a serious zoonotic parasitic disease, is caused by the larvae of Taenia solium and has been acknowledged by the World Organization for Animal Health. The current detection methods of Cysticercus cellulosae cannot meet the needs of large-scale and rapid detection in the field. We hypothesized that the immunofluorescence chromatography test strip (ICS) for detecting Cysticercus cellulosae, according to optimization of a series of reaction systems was conducted, and sensitivity, specificity, and stability testing, and was finally compared with ELISA. This method utilizes Eu3+-labeled time-resolved fluorescent microspheres (TRFM) coupled with TSOL18 antigen to detect TSOL18 antibodies in infected pig sera. RESULTS: ICS and autopsy have highly consistent diagnostic results (n = 133), as determined by Cohen's κ analysis (κ = 0.925). And the results showed that the proposed ICS are high sensitivity (0.9459) with specificity (0.9792). The ICS was unable to detect positive samples of other parasites. It can be stored for at least six months at 4℃. CONCLUSIONS: In summary, we established a TRFM-ICS method with higher sensitivity and specificity than indirect ELISA. Results obtained from serum samples can be read within 10 min, indicating a rapid, user-friendly test suitable for large-scale field detection.

A novel immunofluorescent test system for SARS-CoV-2 detection in infected cells.

Highly variable pandemic coronavirus SARS-CoV-2, which causes the hazardous COVID-19 infection, has been persistent in the human population since late 2019. A prompt assessment of individual and herd immunity against the infection can be accomplished by using rapid tests to determine antiviral antibody levels. The microneutralization assay (MN) is one of the most widely used diagnostic methods that has been proposed to assess the qualitative and quantitative characteristics of virus-specific humoral immunity in COVID-19 convalescents or vaccine recipients. However, some aspects of the assay, such as sensitivity and time cost, need improvement. Here, we developed an express test, which may be potentially used in clinical practice for the assessment of serum-caused SARS-CoV-2 inhibition in infected cell cultures. It implies the detection and counting of coronaviral fluorescent-forming units (FFU) and includes two sequentially used developing components: biotinylated mouse monoclonal antibodies against the recombinant N protein of SARS-CoV-2 (B.1) and the recombinant EGFP-streptavidin fusion protein. Due to the universal specificity of the antibodies, our analytical tool is suitable for the detection of various strains of SARS-CoV-2 when determining both the infectious titer of viruses and the titer of serum virus-neutralizing antibodies. The developed two-component test system is characterized by high sensitivity, a reduced number of analytic stages and low assay cost, as well as by flexibility, since it may be modified for detection of other pathogens using the appropriate antibodies.

Citizen science in action: Time-resolved immunofluorescence-based field detection of antibiotics with portable analytical kit.

Citizen scientist-based environmental monitoring and public education are becoming increasingly popular. However, current technologies for antibiotic-based novel contaminant identification are still restricted to laboratory sample collection and analysis due to detection methodologies and apparatus limitations. This study developed a time-resolved immunofluorescence-based simultaneous field-based assay for ciprofloxacin (CIP) and enrofloxacin (ENR) that matches test results to geographic locations. The assay helps the public understand the potential levels of antibiotic exposures in their environments and helps them take appropriate action to reduce risk. The assay was developed using smartphones and social software in addition to rapid testing. The method uses a portable, low-cost analytical kit with a smartphone app to build a field-based detection platform for the detection and analysis of ENR and CIP in water and aquatic products. The methodological evaluation was good, with detection limits of 0.4 ng/mL and 0.5 ng/g for ENR in water and fish, and quantification limits of 1.2 ng/mL and 1.4 ng/g, with recoveries of 89.0 %-101.0 % and 78.0 %-97.0 %. For CIP in water and fish, the limits of detection were 0.3 ng/mL and 0.4 ng/g, the limits of quantification were 0.9 ng/mL and 1.2 ng/g, and the recoveries were 75.0 %-91.0 % and 72.0 %-89.0 %, both with coefficients of variation <15 %. These limits were sufficient to prevent the two antibiotics from crossing over during simultaneous detection. The assay was validated using real samples to assess the effectiveness of the assay platform in field deployments, and the results were consistent with those obtained through liquid chromatography-tandem mass spectrometry (LC-MS) and enzyme-linked immunoassay (ELISA) techniques. In addition, the TRFIA assay process requires less time, uses more portable instruments, and is less complex than traditional methods. This study provides a new scientific, accurate, and rapid detection method for antibiotic detection by citizen scientists, helping scientists to obtain a wider range of data and providing more opportunities to solve scientific problems.

Preparation and application of fluorescent monoclonal antibodies recognizing goat CD4+CD25+ regulatory T cells.

Regulatory T cells (Tregs) are a subset of T cells participating in a variety of diseases including mycoplasmal pneumonia, contagious ecthyma, and so on. The role of Tregs in goat contagious ecthyma is not completely understood due to the lack of species-specific antibodies. Here, we developed a combination of CD4 and CD25 fluorescence monoclonal antibodies (mAb) to recognize goat Tregs and assessed its utility in flow cytometry, immunofluorescence staining. Using immunofluorescence staining, we found that the frequency of Treg cells was positively correlated with the viral load during orf virus infection. These antibodies could serve as important tools to monitor Tregs during orf virus infection in goats. KEY POINTS: • A combination of fluorescent mAbs (C11 and D12) was prepared for the detection of goat Tregs. • C11 and D12 are effective in flow cytometry, immunofluorescence staining, and C11 has excellent species specificity. • The frequency of Treg cells was positively correlated with the viral load during orf virus infection.

A correlated light and electron microscopy approach to study the subcellular localization of phosphorylated vimentin in human lung tissue.

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence light and electron microscopy. Here, we describe a fast and simple method for correlative immunofluorescence and immunogold labeling on the same section to elucidate the localization of phosphorylated vimentin (P-Vim), a robust feature of pulmonary vascular remodeling in cells of human lung small arteries. The lung is a complex, soft and difficult tissue to prepare for transmission electron microscopy (TEM). Detailing the molecular composition of small pulmonary arteries (<500µm) would be of great significance for research and diagnostics. Using the classical methods of immunochemistry (either hydrophilic resin or thin cryosections), is difficult to locate small arteries for analysis by TEM. To address this problem and to observe the same structures by both light and electron microscopy, correlative microscopy is a reliable approach. Immunofluorescence enables us to know the distribution of P-Vim in cells but does not provide ultrastructural detail on its localization. Labeled structures selected by fluorescence microscope can be identified and further analyzed by TEM at high resolution. With our method, the morphology of the arteries is well preserved, enabling the localization of P-Vim inside pulmonary endothelial cells. By applying this approach, fluorescent signals can be directly correlated to the corresponding subcellular structures in areas of interest.

MIM-CyCIF: masked imaging modeling for enhancing cyclic immunofluorescence (CyCIF) with panel reduction and imputation.

Cyclic Immunofluorescence (CyCIF) can quantify multiple biomarkers, but panel capacity is limited by technical challenges. We propose a computational panel reduction approach that can impute the information content from 25 markers using only 9 markers, learning co-expression and morphological patterns while concurrently increasing speed and panel content and decreasing cost. We demonstrate strong correlations in predictions and generalizability across breast and colorectal cancer, illustrating applicability of our approach to diverse tissue types.

Comparative Performance Assessment of Novel Fluorescence Immunoassay POCTs for Measuring Circulating Levels of Vitamin-D.

Vitamin D (Vit D) is a fat-soluble molecule acting like a hormone, and it is involved in several biological mechanisms such as gene expression, calcium homeostasis, bone metabolism, immune modulation, viral protection, and neuromuscular functions. Vit D deficiency can lead to chronic hypocalcemia, hyperparathyroidism, and many other pathological conditions; in this context, low and very low levels of 25-hydroxy-vitamin D (25-OH-D) were found to be associated with an increased risk of COVID-19 infection and the likelihood of many severe diseases. For all these reasons, it is important to quantify and monitor 25-OH-D levels to ensure that the serum/blood concentrations are not clinically suboptimal. Serum concentration of 25-OH-D is currently the main indicator of Vit D status, and it is currently performed by different assays, but the most common quantitation techniques involve immunometric methods or chromatography. Nevertheless, other quantitation techniques and instruments are now emerging, such as AFIAS-1® and AFIAS-10® (Boditech and Menarini) based on the immunofluorescence analyzer, that guarantee an automated system with cartridges able to give quick and reliable results as a point-of-care test (POCT). This work aims to compare AFIAS-1® and AFIAS-10® (Boditech and Menarini) Vit D quantitation with Ultra High-Performance Liquid Chromatography coupled with tandem mass spectrometry that currently represents the gold standard technique for Vit D quantitation. The analyses were performed in parallel on 56 samples and in different conditions (from fresh and frozen plasma) to assess the reliability of the results. Any statistically significant differences in methods, the fixed error, and the error proportional to concentration were reported. Results obtained in all conditions showed a good correlation between both AFIAS® instruments and LC-MS/MS, and we can affirm that AFIAS-1® and AFIAS-10® are reliable instruments for measuring 25-OH-D with accuracy and in a fast manner.

Microenvironmental reorganization in brain tumors following radiotherapy and recurrence revealed by hyperplexed immunofluorescence imaging.

The tumor microenvironment plays a crucial role in determining response to treatment. This involves a series of interconnected changes in the cellular landscape, spatial organization, and extracellular matrix composition. However, assessing these alterations simultaneously is challenging from a spatial perspective, due to the limitations of current high-dimensional imaging techniques and the extent of intratumoral heterogeneity over large lesion areas. In this study, we introduce a spatial proteomic workflow termed Hyperplexed Immunofluorescence Imaging (HIFI) that overcomes these limitations. HIFI allows for the simultaneous analysis of > 45 markers in fragile tissue sections at high magnification, using a cost-effective high-throughput workflow. We integrate HIFI with machine learning feature detection, graph-based network analysis, and cluster-based neighborhood analysis to analyze the microenvironment response to radiation therapy in a preclinical model of glioblastoma, and compare this response to a mouse model of breast-to-brain metastasis. Here we show that glioblastomas undergo extensive spatial reorganization of immune cell populations and structural architecture in response to treatment, while brain metastases show no comparable reorganization. Our integrated spatial analyses reveal highly divergent responses to radiation therapy between brain tumor models, despite equivalent radiotherapy benefit.

Comparison of the Capacity of Several Machine Learning Tools to Assist Immunofluorescence-Based Detection of Anti-Neutrophil Cytoplasmic Antibodies.

The success of artificial intelligence and machine learning is an incentive to develop new algorithms to increase the rapidity and reliability of medical diagnosis. Here we compared different strategies aimed at processing microscope images used to detect anti-neutrophil cytoplasmic antibodies, an important vasculitis marker: (i) basic classifier methods (logistic regression, k-nearest neighbors and decision tree) were used to process custom-made indices derived from immunofluorescence images yielded by 137 sera. (ii) These methods were combined with dimensional reduction to analyze 1733 individual cell images. (iii) More complex models based on neural networks were used to analyze the same dataset. The efficiency of discriminating between positive and negative samples and different fluorescence patterns was quantified with Rand-type accuracy index, kappa index and ROC curve. It is concluded that basic models trained on a limited dataset allowed for positive/negative discrimination with an efficiency comparable to that obtained by conventional analysis performed by humans (0.84 kappa score). More extensive datasets and more sophisticated models may be required for efficient discrimination between fluorescence patterns generated by different auto-antibody species.

Prolonged exposure to lung-derived cytokines is associated with activation of microglia in patients with COVID-19.

BACKGROUNDSurvivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung-derived pro-inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans.METHODSWe measured levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from 341 patients with respiratory failure and 13 healthy controls, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We used flow cytometry to sort neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing.RESULTSMicroglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro-inflammatory cytokines. Peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19-specific cytokines.CONCLUSIONProlonged lung inflammation results in sustained elevations in circulating cytokines in patients with SARS-CoV-2 pneumonia compared with those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia-related cognitive dysfunction.FUNDINGSCRIPT U19AI135964, UL1TR001422, P01AG049665, P01HL154998, R01HL149883, R01LM013337, R01HL153122, R01HL147290, R01HL147575, R01HL158139, R01ES034350, R01ES027574, I01CX001777, U01TR003528, R21AG075423, T32AG020506, F31AG071225, T32HL076139.

Obtaining Tissues of Human Amniotic Membrane and Identification of Pluripotent Markers.

The immunofluorescence technique has been used to identify pluripotent markers in the human amniotic epithelial cells (hAEC). hAEC belonging to human fetal membranes, specificamently to amnion layer, and are arising by epiblast, this sugest that the hAEC have characteristics of epiblast cells, in other words, characteristcs of pluripotent stem cells. Here we describe obtaining human amnion tissue and identifying pluripotent markers by immunofluorescence.

RNA and Protein Detection by Single-Molecule Fluorescent in Situ Hybridization (smFISH) Combined with Immunofluorescence in the Budding Yeast S. cerevisiae.

The inherent stochastic processes governing gene expression give rise to heterogeneity across individual cells, highlighting the importance of single-cell studies. The emergence of single-molecule fluorescent in situ hybridization (smFISH) enabled gene expression analysis at the single-cell level while including the spatial dimension through the visualization and quantification of mRNAs in intact fixed cells. By combining smFISH with immunofluorescence (IF), a comprehensive approach takes shape facilitating the study of mRNAs and proteins to correlate gene expression profiles to different cellular states. This chapter serves as a comprehensive guide to a smFISH-IF protocol optimized for gene expression analysis in the budding yeast S. cerevisiae. We utilize smFISH to visualize the mRNA localization pattern of the CLB2 cyclin over the course of the cell cycle inferred by alpha-tubulin IF.

Multiplexed Immunofluorescence and Single-Molecule RNA Fluorescence In Situ Hybridization in Mouse Skeletal Myofibers.

RNA fluorescence in situ hybridization (FISH) is a powerful method to determine the abundance and localization of mRNA molecules in cells. While modern RNA FISH techniques allow quantification at single molecule resolution, most methods are optimized for mammalian cell culture and are not easily applied to in vivo tissue settings. Single-molecule RNA detection in skeletal muscle cells has been particularly challenging due to the thickness and high autofluorescence of adult muscle tissue and a lack of in vitro models for mature muscle cells (myofibers). Here, we present a method for isolation of adult myofibers from mouse skeletal muscle and detection of single mRNA molecules and proteins using multiplexed RNA FISH and immunofluorescence.

Combined 3D DNA FISH, Single-Molecule RNA FISH, and Immunofluorescence.

Nuclear architecture is a potential regulator of gene expression in eukaryotic cells. Studies connecting nuclear architecture to gene expression are often population-averaged and do not report on the cell-level heterogeneity in genome organization and associated gene expression. In this report we present a simple way to combine fluorescence in situ hybridization (FISH)-based detection of DNA, with single-molecule RNA FISH (smFISH) and immunofluorescence (IF), while also preserving the three-dimensional (3D) nuclear architecture of a cell. Recently developed smFISH techniques enable the detection of individual RNA molecules; while using 3D DNA FISH, copy numbers and positions of genes inside the nucleus can be interrogated without interfering with 3D nuclear architecture. Our method to combine 3D DNA FISH with smFISH and IF enables a unique quantitative handle on the central dogma of molecular biology.

Cell-Based Assay to Detect the Autoantibody Serostatus in Patients with Neuromyelitis Optica Spectrum Disorder (NMOSD).

Cell-based assay (CBA) is an immunofluorescence assay that is extensively used for the confirmatory diagnosis of inflammatory demyelinating diseases of the central nervous system, like neuromyelitis optica spectrum disorder (NMOSD). Detecting the type of autoantibody present in the sera of the patients is the primary goal. CBA is the most sensitive and recommended detection method among all similar tools. Briefly, serum autoantibody is screened by transfecting specific cells seeded on cover glasses with full-length specific antigen fused with green fluorescent protein (GFP), followed by treating them with the patient serum used here as the source of primary antibody. The autoantibody-treated cells are further labeled with a rhodamine-conjugated secondary antibody. The co-localization of GFP and rhodamine is visualized by confocal microscopy, and the intensity of fluorescence is evaluated to determine the presence of autoantibody. A detailed protocol to screen antibodies against AQP4 and MOG in human sera using this method is described.

Are telocytes related to maintenance of vascular homeostasis in normal and pathological aorta?

The telocyte (TC) is a new interstitial cell type described in a wide variety of organs and loose connective tissues around small vessels, but its presence in large arteries remains unexplored. TCs have small cell bodies and remarkably thin, long, moniliform processes called telopods (Tps). Using transmission electron microscopy and immunofluorescence, we identified TCs in normal human thoracic aortas and in those with aneurysm or acute dissection (TAAD). In normal aortas the TCs were distributed throughout the connective tissue of the adventitial layer, in its innermost portion and at the zone of transition with the medial layer, with their long axes oriented parallel to the external elastic lamellae, forming a three-dimensional network, without prevalence in the media layer. In contrast, TAAD TCs were present in the medial layer and in regions of neovascularization. The most important feature of the adventitia of diseased aortas was the presence of numerous contacts between TCs and stem cells, including vascular progenitor cells. Although the biologically functional correlations need to be elucidated, the morphological observations presented here provide strong evidence of the involvement of TCs in maintaining vascular homeostasis in pathological situations of tissue injury.