Evaluation of eGFP expression in the ChAT-eGFP transgenic mouse brain.

BACKGROUND: A historically definitive marker for cholinergic neurons is choline acetyltransferase (ChAT), a synthesizing enzyme for acetylcholine, (ACh), which can be found in high concentrations in cholinergic neurons, both in the central and peripheral nervous systems. ChAT, is produced in the body of the neuron, transported to the nerve terminal (where its concentration is highest), and catalyzes the transfer of an acetyl group from the coenzyme acetyl-CoA to choline, yielding ACh. The creation of bacterial artificial chromosome (BAC) transgenic mice that express promoter-specific fluorescent reporter proteins (green fluorescent protein-[GFP]) provided an enormous advantage for neuroscience. Both in vivo and in vitro experimental methods benefited from the transgenic visualization of cholinergic neurons. Mice were created by adding a BAC clone into the ChAT locus, in which enhanced GFP (eGFP) is inserted into exon 3 at the ChAT initiation codon, robustly and supposedly selectively expressing eGFP in all cholinergic neurons and fibers in the central and peripheral nervous systems as well as in non-neuronal cells. METHODS: This project systematically compared the exact distribution of the ChAT-eGFP expressing neurons in the brain with the expression of ChAT by immunohistochemistry using mapping and also made comparisons with in situ hybridization (ISH). RESULTS: We qualitatively described the distribution of ChAT-eGFP neurons in the mouse brain by comparing it with the distribution of immunoreactive neurons and ISH data, paying special attention to areas where the expression did not overlap, such as the cortex, striatum, thalamus and hypothalamus. We found a complete overlap between the transgenic expression of eGFP and the immunohistochemical staining in the areas of the cholinergic basal forebrain. However, in the cortex and hippocampus, we found small neurons that were only labeled with the antibody and not expressed eGFP or vice versa. Most importantly, we found no transgenic expression of eGFP in the lateral dorsal, ventral and dorsomedial tegmental nuclei cholinergic cells. CONCLUSION: While the majority of the forebrain ChAT expression was aligned in the transgenic animals with immunohistochemistry, other areas of interest, such as the brainstem should be considered before choosing this particular transgenic mouse line.

A multi-depth spiral milli fluidic device for whole mount zebrafish antibody staining.

Whole mount zebrafish antibody staining (ABS) is a common staining technique used to localize protein information in a zebrafish embryo or larva. Like most biological assays, the whole mount zebrafish ABS is still largely conducted manually through labor intensive and time-consuming steps which affect both consistency and throughput of the assay. In this work, we develop a milli fluidic device that can automatically trap and immobilize the fixed chorion-less zebrafish embryos for the whole mount ABS. With just a single loading step, the zebrafish embryos can be trapped by the milli fluidic device through a chaotic hydrodynamic trapping process. Moreover, a consistent body orientation (i.e., head point inward) for the trapped zebrafish embryos can be achieved without additional orientation adjustment device. Furthermore, we employed a consumer-grade SLA 3D printer assisted method for device prototyping which is ideal for labs with limited budgets. Notably, the milli fluidic device has enabled the optimization and successful implementation of whole mount zebrafish Caspase-3 ABS. We demonstrated our device can accelerate the overall procedure by reducing at least 50% of washing time in the standard well-plate-based manual procedure. Also, the consistency is improved, and manual steps are reduced using the milli fluidic device. This work fills the gap in the milli fluidic application for whole mount zebrafish immunohistochemistry. We hope the device can be accepted by the zebrafish community and be used for other types of whole mount zebrafish ABS procedures or expanded to more complicated in situ hybridization (ISH) procedure.

GABA Immunoreactivity and Pharmacological Effects vary Among Stylet-Bearing Nematodes.

Plant-parasitic nematodes conduct a series of sophisticated behaviors to complete their life cycles. Among these, locomotion behaviors, including finding the host and migrating to the feeding site, directly affect the success of parasitism. Thus, disrupting locomotion behaviors has the potential to control these parasites. γ-Aminobutyric acid (GABA) is the prominent inhibitory neurotransmitter in nematodes. GABA-immunoreactive neurons are mostly found in motor neurons, where they regulate behaviors in the model nematode C. elegans. However, the GABA system in most stylet-bearing nematodes has received little attention. Using immunohistochemistry, we found variation in the pattern of GABA-immunoreactivity among two major plant-parasites and a fungal feeder. Some of these GABA-immunoreactive neurons lack clear homologs to C. elegans. Pharmaceutical assays showed that applying GABA, its agonist, and its antagonist, can disrupt the locomotion behaviors of these nematodes, although sensitivity to a given compound varied between species. Our data suggest that the GABA system is a potential target for the control of plant-parasitic nematodes.

Staining of antinuclear antibodies and antibodies against removable nuclear antigens in connective tissue diseases.

INTRODUCTION AND OBJECTIVES: Connective tissue diseases are inflammatory, autoimmune diseases and threaten quality of life. To determine the relationship between staining patterns of antinuclear antibodies and antibodies against extractable nuclear antigens in patients with connective tissue disease. MATERIALS AND METHODS: Observational, basic, analytical and transversal study. Study conducted in the Immunology Service of the Arzobispo Loayza National Hospital between January 2017 and June 2017. We analyzed 291 samples of patients with CTD and for the detection of anti-nuclear antibody staining patterns, the immunological kit and observation with microscope of at 40X Immunofluorescence and for the detection of the antibodies against extractable nuclear antigens. The Immunoblot method was employed. Statistical analyses were carried out with the statistical package SPSS version 21 for Windows. We used the Pearson Chi-square test for the categorical variables, a value of p<0.05 was considered significant. RESULTS: There was a significant relationship p<0.05 of the homogeneous pattern, the mottled pattern with Anti-histones (p=0.000), Anti-nucleosomes (p=0.000), Anti-Ro 52 (p=0.000), Anti-SSA (p=0.001), Anti-SSB (p=0.003), Anti-dsDNA (p=0.000) with the Pearson Chi-square test. There was a significant relationship of p<0.05 of the centromeric pattern with Anti-Cenp B (p=0.000) with Fisherâ¿¿s exact statistic. CONCLUSIONS: There was a significant relationship between the anti-nuclear antibody staining patterns and the antibodies to the core extractable antigens in patients with systemic lupus erythematosus, Sjögrenâ¿¿s syndrome, Calcinosis, Raynaudâ¿¿s phenomenon, esophageal Dysmotility, sclerodactyly and Telangiectasia (CREST), Scleroderma and Polymyositis.

Detection of tumor-associated cells in cryopreserved peripheral blood mononuclear cell samples for retrospective analysis.

BACKGROUND: Cryopreserved peripheral blood mononuclear cells (PBMCs) are commonly collected in biobanks. However, little data exist regarding the preservation of tumor-associated cells in cryopreserved collections. The objective of this study was to determine the feasibility of using the CellSieve™ microfiltration assay for the isolation of circulating tumor cells (CTCs) and circulating cancer-associated macrophage-like cells (CAMLs) from cryopreserved PBMC samples. METHODS: Blood samples spiked with breast (MCF-7), prostate (PC-3), and renal (786-O) cancer cell lines were used to establish analytical accuracy, efficiency, and reproducibility after cryopreservation. The spiked samples were processed through Ficoll separation, and cryopreservation was followed by thawing and microfiltration. RESULTS: MCF-7 cells were successfully retrieved with recovery efficiencies of 90.5 % without cryopreservation and 87.8 and 89.0 %, respectively, on day 7 and day 66 following cryopreservation. The corresponding recovery efficiencies of PC-3 cells were 83.3 % without cryopreservation and 85.3 and 84.7 %, respectively, after cryopreservation. Recovery efficiencies of 786-O cells were 92.7 % without cryopreservation, and 82.7 and 81.3 %, respectively, after cryopreservation. The recovered cells retained the morphologic characteristics and immunohistochemical markers that had been observed before freezing. The protocols were further validated by quantitation of CAMLs in blood samples from two patients with renal cell carcinoma (RCC). The recovery rates of CTCs and CAMLs from cryopreserved samples were not statistically significant different (P > 0.05) from matched fresh samples. CONCLUSIONS: To our knowledge, this is the first report that CAMLs could be cryopreserved and analyzed after thawing with microfiltration technology. The application of microfiltration technology to cryopreserved samples will enable much greater retrospective study of cancer patients in relation to long-term outcomes.

Validation of CSF-1 receptor (CD115) staining for analysis of murine monocytes by flow cytometry.

CD115, the receptor for colony stimulating factor 1, is essential for survival and differentiation of monocytes and macrophages and is therefore frequently used to define monocyte subsets and their progenitors in immunological assays. However, CD115 surface expression and detection by flow cytometry is greatly influenced by cell isolation and processing methods, organ source, and disease context. In a systematic analysis of murine monocytes, we define experimental conditions that preserve or limit CD115 surface expression and staining by flow cytometry. We also find that, independent of conditions, CD115 surface levels are consistently lower in Ly6Clo monocytes than in Ly6Chi monocytes, with the exception of Ly6Clo monocytes in the bone marrow. Furthermore, in contrast to IL-34, the presence of colony stimulating factor 1 impairs CD115 antibody staining in a dose-dependent manner, which, in a model of ischemic kidney injury with elevated levels of colony stimulating factor 1, influenced quantification of kidney monocytes. Thus, staining and experimental conditions affect quantitative and qualitative analysis of monocytes and may influence experimental conclusions.

Detection of West Nile Virus Envelope Protein in Brain Tissue with an Immunohistochemical Assay.

Immunohistochemistry is a valuable tool for probing not only scientific questions but also clinical diagnoses. It provides power from localization of a protein within the milieu of a tissue section that may reflect positioning within or beyond the boundaries of a cell that is representative of the tissue at a discrete moment in time. The method can be applied broadly, including to tissues under normal, developmental, chemically, or genetically altered conditions and disease states.Disease manifesting from West Nile virus infection ranges from acute, systemic febrile symptoms to compromise of central nervous system function. Immunohistochemistry has been used to assess WNV infection in the nervous system in postmortem and experimental conditions, despite the lack of understanding of the precise route of viral entry. In addition to imprecise knowledge of initial viral entry into cells and whether entry is even the same between cell types, the fact that spontaneous viral mutations and environmental pressures from climate change may alter the prevalence of the disease state across geographical and climatological boundaries highlights the need for continued assessment of infection. Immunohistochemistry is a useful way to assess these aspects of WNV infection with the aim being to better understand the organs and cell types that are compromised by WNV infection. This chapter outlines how this can be carried out on brain tissue, but the procedures discussed can also be applied more broadly on tissue outside of the central nervous system.

Flow Cytometry Analysis of Immune Cell Responses.

Flow cytometry is a fluorescence-based technology that allows for the identification and characterization of immune cell subsets within a heterogenous population. Briefly, isolated immune cells are stained in suspension with fluorescently tagged antibodies to identify cells of interest prior to being run through a flow cytometer. Here we describe how to isolate murine immune cells from various body regions, including the inguinal lymph nodes (ILNs), spleen, thymus, and peripheral blood, and tag them with primary fluorescent antibodies for flow cytometric analysis of CD4+ and CD8+ T cell populations. This chapter also details how to use flow cytometry to measure T cell expression of chemokine receptor 7 (CCR7), the major chemokine receptor lymphocytes use to enter lymph nodes. The methods described in this chapter can be used for characterizing other proteins of interest, as well as other immune cell subsets.

Ethyl Cinnamate-Based Tissue Clearing Strategies.

Tissue clearing turns otherwise turbid and opaque tissue transparent, enabling imaging deep within tissues. The nontransparent nature of most tissues is due to the refractive index mismatch between its three major constituent components (lipids, proteins, and water). All tissue clearing methods rectify this mismatch by homogenizing the refractive index within the tissue and carefully matching it to the surrounding media. Here we describe a detailed protocol to clear a wide range of salamander tissues. We also include several optional steps such as depigmentation, antibody staining, and tissue mounting. These steps are optional, and do not change anything in the steps needed for tissue clearing. Depending on the fluorescent signal and optics employed, images up to several millimeters inside of the tissue can be acquired.

A Simplified Approach to Evaluating T Cell Development by Flow Cytometry.

T cell development is a complex multistep process that requires the coordinated activation of distinct signaling responses and the regulated progression of developing cells (thymocytes) through key stages of maturation. Although sophisticated techniques such as fetal thymus organ culture, in vitro thymocyte culture, and multiparameter flow cytometry-based cell sorting are now widely employed to evaluate thymocyte maturation by experienced laboratories, defects in T cell development can usually be identified with relatively simple flow cytometry screening methods. Here, we provide a basic protocol for assessment of T cell development that will enable laboratories with access to a multi-laser flow cytometer to screen mouse strains, including those generated from embryonic stem cells with targeted gene mutations, for thymocyte maturation defects.

Dissecting Molecular Phenotypes Through FACS-Based Pooled CRISPR Screens.

Pooled CRISPR screens are emerging as a powerful tool to dissect regulatory networks, by assessing how a protein responds to genetic perturbations in a highly multiplexed manner. A large number of genes are perturbed in a cell population through genomic integration of one single-guide RNA (sgRNA) per cell. A subset of cells with the phenotype of interest can then be enriched through fluorescence-activated cell sorting (FACS). SgRNAs with altered abundance after phenotypic enrichment allow identification of genes that either promote or attenuate the investigated phenotype. Here we provide detailed guidelines on how to design and execute a pooled CRISPR screen to investigate molecular phenotypes. We describe how to generate a custom sgRNA library and how to perform a FACS-based screen using readouts such as intracellular antibody staining or Flow-FISH to assess phosphorylation levels or RNA abundance. Through the variety of available perturbation systems and readout options many different molecular and cellular phenotypes can now be tackled with pooled CRISPR screens.

Experimental Methodologies to Visualize Early Cell Biology in Zebrafish Embryos.

For organisms to function normally, biological molecules must work at the correct time in the right cells and within the right intracellular compartments of cells. Biological research relies heavily on discovering the cellular locations at which such molecular interactions occur. A mainstay technique in this process of discovery is the visualization of locations of proteins in cells and tissues, known as immunocytochemistry and immunohistochemistry, respectively. If performed correctly, these techniques can provide detailed information regarding the endogenous locations of proteins and their ectopic locations or absence in mutants and in disease states.

Anatomical Comparison of Antennal Lobes in Two Sibling Ectropis Moths: Emphasis on the Macroglomerular Complex.

Ectropis obliqua and Ectropis grisescens are two sibling moth species of tea plantations in China. The male antennae of both species can detect shared and specific sex pheromone components. Thus, the primary olfactory center, i.e., the antennal lobe (AL), plays a vital role in distinguishing the sex pheromones. To provide evidence for the possible mechanism allowing this distinction, in this study, we compared the macroglomerular complex (MGC) of the AL between the males of the two species by immunostaining using presynaptic antibody and propidium iodide (PI) with antennal backfills, and confocal imaging and digital 3D-reconstruction. The results showed that MGC of both E. obliqua and E. grisescens contained five glomeruli at invariant positions between the species. However, the volumes of the anterior-lateral glomerulus (ALG) and posterior-ventral (PV) glomerulus differed between the species, possibly related to differences in sensing sex pheromone compounds and their ratios between E. obliqua and E. grisescens. Our results provide an important basis for the mechanism of mating isolation between these sibling moth species.

Xanthogranulomatous appendicitis: A comprehensive literature review.

BACKGROUND: Xanthogranulomatous inflammation is characterized histologically by a collection of lipid-laden macrophages admixed with lymphocytes, plasma cells, neutrophils, and often multinucleated giant cells with or without cholesterol clefts. AIM: To review the medical literature on xanthogranulomatous appendicitis (XGA). METHODS: We present a patient with XGA and review published articles on XGA accessed via the PubMed, MEDLINE, Google Scholar, and Google databases. Keywords used were "appendix vermiformis," "appendectomy," "acute appendicitis," and "XGA." The search included articles published before May 2020, and the publication language was not restricted. The search included letters to the editor, case reports, review articles, original articles, and meeting presentations. Articles or abstracts containing adequate information about age, sex, clinical presentation, white blood cells, initial diagnosis, surgical approach, histopathological and immunohistochemical features of appendectomy specimens were included in the study. RESULTS: A total of 29 articles involving 38 patients with XGA, were retrospectively analyzed. Twenty (52.6%) of the 38 patients, aged 3 to 78 years (median: 34; IQR: 31) were female, and the remaining 18 (47.4%) were male. Twenty-five patients were diagnosed with acute appendicitis, ruptured appendicitis, or subacute appendicitis, and the remaining 13 patients underwent surgery for tumoral lesions of the ileocecal region. Twenty-two of the patients underwent urgent or semi-urgent surgery, and the remaining 16 patients underwent interval appendectomy. CONCLUSION: Xanthogranulomatous inflammation rarely affects the appendix vermiformis. It is associated with significant diagnostic and therapeutic dilemmas due to its variable presentation. It is often associated with interval appendectomies, and a significant number of patients require bowel resection due to the common presentation of a tumoral lesion. XGA is usually identified retrospectively on surgical pathology and has no unique features in preoperative diagnostic studies.

The Application of Flow Cytometry for Simultaneous and Multi-parametric Analysis of Heterogenous Cell Populations in Basic and Clinical Research.

The use of flow cytometry allows simultaneous measurement and multiparametric analysis of single cells in a heterogenous solution. The purpose of flow cytometry can vary depending on the use of antibodies and dyes targeted for specific cell molecules. The method of immune-phenotyping with fluorescently conjugated antibodies to label cell proteins or DNA works in tandem with fluidic, optic, and electrical systems present in the flow cytometer. Some flow cytometers can detect numerous fluorescent molecules on a single cell, allowing the measurement of more than 30 parameters. This ability to detect, measure, and quantitate multiple fluorescent markers on a single cell makes the flow cytometer a useful tool for analyzing various aspects of cell phenotype and function. Here we describe a standardized protocol for surface and intracellular immune-phenotyping of murine lungs, beginning with the building of an optimal antibody panel and ending with data analysis and representation, including sample gating strategies for innate and adaptive immune responses.

Staining MIF in Cells for Confocal Microscopy.

Confocal microscopy is a powerful technique for immunofluorescence imaging of cells and tissues. The technique allows for detailed analysis of intracellular localization of molecules, as well as three-dimensional representation and analysis of samples, and can be used as a gateway to more advanced techniques, including FLIM-FRET and super-resolution microscopy. Relatively few studies have used confocal microscopy to study intracellular localization of macrophage migration inhibitory factor (MIF) in detail. This chapter outlines basic protocols and tips for staining MIF in fixed cells for confocal analysis.

Immunostainings in Nervous System Development of the Nematode C. elegans.

The nematode C. elegans is a useful model organism for studying neuronal development and function due to its extremely simple, well-defined nervous system, translucence, short life cycle, and abundance of genetic tools (WormBase. http://wormbase.org , 2018; WormBook. The C. elegans Research Community. http://www.wormbook.org , 2018). Due to the relative ease of genetic transformation, the majority of studies in C. elegans use transgenes (e.g., green fluorescent proteins) to assess the expression and distribution of specific proteins. In addition, large-scale in situ hybridization studies have described the distribution of mRNAs for thousands of genes throughout development. However, there may be qualitative and quantitative differences between expression of transgenic markers and the endogenous protein. Specific antibodies can be difficult to generate, but once generated antibodies can be used to study protein function and changes in expression and localization during development. Thus, genetic tools and immunohistochemistry are complementary techniques for studying cellular and developmental processes in C. elegans. Protocols for immunostaining in C. elegans are similar to those in other organisms; however, some features of these nematodes provide unique challenges. These include difficulties with antibody generation and access to the nervous system through the cuticle. This chapter describes a basic immunostaining technique that works in C. elegans for a variety of neural antigens in all stages of development to use in conjunction with the many tools available in this simple animal.

Whole-mount Immunohistochemistry of Adult Zebrafish Retina for Advanced Imaging.

Immunohistochemistry is a widely used technique to examine the expression and subcellular localization of proteins. This technique relies on the specificity of antibodies and requires adequate penetration of antibodies into tissues. The latter is especially challenging for thick specimens, such as embryos and other whole-mount preparations. Here we describe an improved method of immunohistochemistry for retinal whole-mount preparations. We report that a cocktail of three reagents, Triton X-100, Tween-20, and DMSO, in blocking and antibody dilution buffers strongly enhances immunolabeling in whole-mount retinas from adult zebrafish. In addition, we establish that in whole retinal tissues, a classic epitope retrieval method, based on citrate buffer, is effective for immunolabeling membrane-associated proteins. Overall, this simple modification allows precise and reproducible immunolabeling of proteins in retinal whole-mounts.

Nonfluorescent RNA In Situ Hybridization Combined with Antibody Staining to Visualize Multiple Gene Expression Patterns in the Embryonic Brain of Drosophila.

In Drosophila, the brain arises from about 100 neural stem cells (called neuroblasts) per hemisphere which originate from the neuroectoderm. Products of developmental control genes are expressed in spatially restricted domains in the neuroectoderm and provide positional cues that determine the formation and identity of neuroblasts. Here, we present a protocol for nonfluorescent double in situ hybridization combined with antibody staining which allows the simultaneous representation of gene expression patterns in Drosophila embryos in up to three different colors. Such visible multiple stainings are especially useful to analyze the expression and regulatory interactions of developmental control genes during early embryonic brain development. We also provide protocols for wholemount and flat preparations of Drosophila embryos, which allow a more detailed analysis of gene expression patterns in relation to the cellular context of the early brain (and facilitate the identification of individual brain neuroblasts) using conventional light microscopy.

Analysis of Complete Neuroblast Cell Lineages in the Drosophila Embryonic Brain via DiI Labeling.

Proper functioning of the brain relies on an enormous diversity of neural cells generated by neural stem cell-like neuroblasts (NBs). Each of the about 100 NBs in each side of brain generates a nearly invariant and unique cell lineage, consisting of specific neural cell types that develop in defined time periods. In this chapter we describe a method that labels entire NB lineages in the embryonic brain. Clonal DiI labeling allows us to follow the development of an NB lineage starting from the neuroectodermal precursor cell up to the fully developed cell clone in the first larval instar brain. We also show how to ablate individual cells within an NB clone, which reveals information about the temporal succession in which daughter cells are generated. Finally, we describe how to combine clonal DiI labeling with fluorescent antibody staining that permits relating protein expression to individual cells within a labeled NB lineage. These protocols make it feasible to uncover precise lineage relationships between a brain NB and its daughter cells, and to assign gene expression to individual clonal cells. Such lineage-based information is a critical key for understanding the cellular and molecular mechanisms that underlie specification of cell fates in spatial and temporal dimension in the embryonic brain.