Superresolution Expansion Microscopy in Dictyostelium Amoebae.

Expansion microscopy (ExM) is a superresolution technique for fixed specimens that improves resolution of a given microscopy system approximately fourfold. The gain in resolution in ExM is not achieved by improvement of the resolution of the microscope itself but by isotropic expansion of the sample. To achieve this, the sample is cross-linked to an expandable gel matrix that swells approximately fourfold by incubation in water. We have applied the method to Dictyostelium amoebae and discuss the pros and cons of different labeling techniques in combination with pre- and post-expansion staining protocols.

APEX2-Mediated Proximity Protein Labeling in Dictyostelium.

Largely due to its simplicity, while being more like human cells compared to other experimental models, Dictyostelium continues to be of great use to discover basic molecular mechanisms and signaling pathways underlying evolutionarily conserved biological processes. However, the identification of new protein interactions implicated in signaling pathways can be particularly challenging in Dictyostelium due to its extremely fast signaling kinetics coupled with the dynamic nature of signaling protein interactions. Recently, the proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells was shown to allow the detection of weak and/or transient protein interactions and also to obtain spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in Dictyostelium. Coupled with the identification of the labeled proteins by mass spectrometry, this method expands Dictyostelium's proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in Dictyostelium.

[Imaging for displaying lymphatic vessels in whole-mount aorta adventitia of ApoE-/- mice by immunofluorescent histochemical staining].

Objective To explore a simple and feasible method for whole-mount immunofluorescence staining of lymphatic vessels in the ApoE-/- mouse model of atherosclerosis. Methods Aortic specimens were carefully excised from the ApoE-/- mouse model. Following immunostaining with specific antibodies against smooth muscle actin (SMA) and lymphatic vessel endothelial receptor 1 (LYVE1), the aortas, including the aortic root, were subjected to a 30-minute treatment with 5 g/L Sudan Black B solution. This step was instrumental in minimizing the autofluorescent background of the tissue. Thereafter, the aortas were processed through a clearing protocol and imaged within a purpose-built chamber under a fluorescence microscope. Results The pretreatment with 5 g/L Sudan Black B effectively suppressed the autofluorescent signals emanating from the vascular structures, thereby enhancing the contrast and clarity of the specific fluorescence signals associated with the lymphatic vessels. This enhancement in signal quality did not compromise the integrity or specificity of the immunofluorescent markers. Conclusion A facile, highly specific, and effective approach for the visualization of lymphatic vessels in whole-mount aortic preparations from ApoE-/- mice is established.

Tetranuclear Polypyridylruthenium(II) Complexes as Selective Nucleic Acid Stains for Flow Cytometric Analysis of Monocytic and Epithelial Lung Carcinoma Large Extracellular Vesicles.

Selective staining of extracellular vesicles (EVs) is a major challenge for diagnostic and therapeutic applications. Herein, the EV labeling properties of a new class of tetranuclear polypyridylruthenium(II) complexes, Rubb7-TNL and Rubb7-TL, as phosphorescent stains are described. These new stains have many advantages over standard stains to detect and characterize EVs, including: high specificity for EV staining versus cell staining; high phosphorescence yields; photostability; and a lack of leaching from EVs until incorporation with target cells. As an example of their utility, large EVs released from control (basal) or lipopolysaccharide (LPS)-stimulated THP-1 monocytic leukemia cells were studied as a model of immune system EVs released during bacterial infection. Key findings from EV staining combined with flow cytometry were as follows: (i) LPS-stimulated THP-1 cells generated significantly larger and more numerous large EVs, as compared with those from unstimulated cells; (ii) EVs retained native EV physical properties after staining; and (iii) the new stains selectively differentiated intact large EVs from artificial liposomes, which are models of cell membrane fragments or other lipid-containing debris, as well as distinguished two distinct subpopulations of monocytic EVs within the same experiment, as a result of biochemical differences between unstimulated and LPS-stimulated monocytes. Comparatively, the staining patterns of A549 epithelial lung carcinoma-derived EVs closely resembled those of THP-1 cell line-derived EVs, which highlighted similarities in their selective staining despite their distinct cellular origins. This is consistent with the hypothesis that these new phosphorescent stains target RNA within the EVs.

Automated neuronal reconstruction with super-multicolour Tetbow labelling and threshold-based clustering of colour hues.

Fluorescence imaging is widely used for the mesoscopic mapping of neuronal connectivity. However, neurite reconstruction is challenging, especially when neurons are densely labelled. Here, we report a strategy for the fully automated reconstruction of densely labelled neuronal circuits. Firstly, we establish stochastic super-multicolour labelling with up to seven different fluorescent proteins using the Tetbow method. With this method, each neuron is labelled with a unique combination of fluorescent proteins, which are then imaged and separated by linear unmixing. We also establish an automated neurite reconstruction pipeline based on the quantitative analysis of multiple dyes (QDyeFinder), which identifies neurite fragments with similar colour combinations. To classify colour combinations, we develop unsupervised clustering algorithm, dCrawler, in which data points in multi-dimensional space are clustered based on a given threshold distance. Our strategy allows the reconstruction of neurites for up to hundreds of neurons at the millimetre scale without using their physical continuity.

Performance of fine needle aspiration cytology and Ziehl-Neelsen staining technique in the diagnosis of tuberculosis lymphadenitis.

INTRODUCTION: Proper diagnosis of tuberculosis (TB) lymphadenitis is critical for its treatment and prevention. Fine needle aspirate cytology (FNAC) is the mainstay method for the diagnosis of TB lymphadenitis in Ethiopia; however, the performance of FNAC has not been evaluated in the Eastern Region of Ethiopia. This study aimed to evaluate the performance of FNAC and Ziehl-Neelsen (ZN) staining compared with that of GeneXpert for the diagnosis of TB lymphadenitis. METHODS: Fine needle aspiration (FNA) specimens collected from 291 patients suspected of having TB lymphadenitis were examined using FNAC, ZN, and GeneXpert to diagnose TB lymphadenitis. Gene-Xpert was considered the reference standard method for comparison. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and kappa coefficient were determined using SPSS version 25. RESULTS: The sensitivity, specificity, PPV, and NPV of ZN for diagnosing TB lymphadenitis were 73.2%, 97.4%, 96.2%, and 80.1% respectively. There was poor agreement between ZN and GeneXpert (Kappa=-0.253). The sensitivity, specificity, PPV, and NPV of FNAC were 83.3%, 94.8%, 93.5%, and 86.3% respectively. There was moderate agreement between the FNAC and GeneXpert (Kappa = 0.785). CONCLUSION: The fine needle aspiration cytology (FNAC) is a more sensitive test for the diagnosis of TB lymphadenitis than ZN. The FNAC showed a moderate agreement with the GeneXpert assay. This study recommends the FNA GeneXpert MTB/RIF test in preference to FNAC for the diagnosis of TB lymphadenitis to avoid a missed diagnosis of smear-negative TB lymphadenitis.

Significance of Various Diagnostic Modalities in Detection of Tuberculosis in Cervical Lymphadenopathy: A Study of 200 Cases.

BACKGROUND: Extrapulmonary tuberculosis (EPTB) makes for 25% of all instances of tuberculosis (TB) patients. The enigmatic clinical presentation of EPTB makes identification difficult since it simulates other chronic conditions such as neoplastic and inflammatory disorders and could culminate in treatment that is either insufficient or not required. For an affirmative and confirmed diagnosis, a substantial level of suspicion is imperative. The paucibacillary feature of EPTB makes diagnosis extremely difficult and necessitates the use of many diagnostic methods to arrive at a precise diagnosis. In December 2010, the World Health Organization recommended using GeneXpert/cartridge-based nucleic acid amplification test (CBNAAT) for the initial assessment of suspected cases of EPTB. Furthermore, fine-needle aspiration cytology (FNAC), Ziehl-Neelsen (ZN) stain, and the CBNAAT have to be utilized to exclude other possible origins of granulomatous inflammation. The goal of the current investigation is to comprehend how FNAC and ZN stains relate to CBNAAT and their diagnostic value. METHODS: The evaluation included all suspected instances of tubercular lymphadenopathy, and adequate aspirates were obtained from the site of the enlarged cervical lymph nodes. Smears were made following FNAC and stained with ZN stain as well as hematoxylin and eosin stain. Simultaneously, CBNAAT and culture evaluations were conducted on the same aspirates. This cross-sectional study took place at a tertiary care center and encompassed 200 individuals with clinical manifestations of EPTB. RESULTS: There were 200 cases of suspected tubercular lymphadenitis (TBLN). According to the FNAC results, TBLN was detected in 71 (47.6%) of these 200 cases, followed by necrotizing lymphadenitis in 56 (37.5%), chronic caseating granulomatous lymphadenitis in 47 (31.5%), and reactive lymphadenitis in 26 (17.4%). They were correlated with CBNAAT results, which showed that all instances of tuberculous lymphadenitis, 85.71% of cases of necrotizing lymphadenitis, 55.32% of cases of chronic caseating granulomatous lymphadenitis, and 2 (7.69%) cases of reactive lymphadenitis were CBNAAT positive. CONCLUSION: CBNAAT should be utilized with FNAC and ZN staining to diagnose EPTB. The CBNAAT assay demonstrated a significant advantage in the identification of previously unidentified FNAC patients. Despite being a simple diagnostic tool, FNAC has a lower specificity and significantly lower precision than CBNAAT in correctly identifying cases of EPTB because it exhibits similar cytomorphological characteristics with lesions that are not associated with TB.

Qualitative and quantitative analysis of lipid droplets in mature 3T3-L1 adipocytes using oil red O.

By differentiating into mature adipocytes, 3T3-L1 cells can be utilized as a model cell line to investigate (pre)adipocyte function in vitro. Here, we present a protocol for combining qualitative and quantitative analysis of lipid droplets in mature 3T3-L1 adipocytes using oil red O. We describe steps to differentiate 3T3-L1 preadipocytes to adipocytes and give detailed procedures to determine total lipid amount as well as lipid droplet size and number using microscopic devices and an ImageJ macro. For complete details on the use and execution of this protocol, please refer to Kaczmarek et al.1.

Quantitative Analysis of Phagocytosis in Whole Blood Using Double Staining and Visualization.

Phagocytosis is an essential innate immunity function in humans and animals. A decrease in the ability to phagocytize is associated with many diseases and aging of the immune system. Assessment of phagocytosis dynamics requires quantification of bacteria inside and outside the phagocyte. Although flow cytometry is the most common method for assessing phagocytosis, it does not include visualization and direct quantification of location of bacteria. Here, we used double-labeled Escherichia coli cells to evaluate phagocytosis by flow cytometry (cell sorting) and confocal microscopy, as well as employed image cytometry to provide high-throughput quantitative and spatial recognition of the double-labeled E. coli associated with the phagocytes. Retention of pathogens on the surface of myeloid and lymphoid cells without their internalization was suggested to be an auxiliary function of innate immunity in the fight against infections. The developed method of bacterial labeling significantly increased the accuracy of spatial and quantitative measurement of phagocytosis in whole blood and can be recommended as a tool for phagocytosis assessment by image cytometry.

Two protocols for the detection of oleaginous bacteria using Oil Red O.

The selection of oleaginous bacteria, potentially applicable to biotechnological approaches, is usually carried out by different expensive and time-consuming techniques. In this study, we used Oil Red O (ORO) as an useful dye for staining of neutral lipids (triacylglycerols and wax esters) on thin-layer chromatography plates. ORO could detect minimal quantities of both compounds (detection limit, 0.0025 mg of tripalmitin or 0.005 mg of cetylpalmitate). In addition, we developed a specific, rapid, and inexpensive screening methodology to detect triacylglycerol-accumulating microorganisms grown on the agar plate. This staining methodology detected 9/13 strains with a triacylglycerol content higher than 20% by cellular dry weight. ORO did not stain polyhydroxyalkanoates-producing bacteria. The four oleaginous strains not detected by this screening methodology exhibited a mucoid morphology of their colonies. Apparently, an extracellular polymeric substance produced by these strains hampered the entry of the lipophilic dye into cells. The utilization of the developed screening methodology would allow selecting of oleaginous bacteria in a simpler and faster way than techniques usually used nowadays, based on unspecific staining protocols and spectrophotometric or chromatographic methods. Furthermore, the use of ORO as a staining reagent would easily characterize the neutral lipids accumulated by microorganisms as reserve compounds. KEY POINTS: • Oil Red O staining is specific for triacylglycerols • Oil Red O staining is useful to detect oleaginous bacteria • Fast and inexpensive staining to isolate oleaginous bacteria from the environment.

Comparison of staining quality between rapid and routine hematoxylin and eosin staining of frozen breast tissue sections: an observational study.

OBJECTIVE: To compare the staining quality between rapid hematoxylin and eosin (H&E) staining and routine H&E staining of frozen breast tissue sections. METHODS: In this cross-sectional observational study, 120 frozen breast tissue sections were randomly assigned to rapid or routine H&E staining (n = 60 per group). Rapid H&E staining used a 7:1 mixture of modified Gill's hematoxylin and alcohol-soluble 1% eosin Y. The staining quality of each section was evaluated and scored. A score of >7 was considered excellent, a score of 6 to 7 good, and a score of ≤5 poor. RESULTS: The staining time for rapid staining was approximately 3 minutes, whereas that of routine staining was approximately 12 minutes. There were no significant differences in the staining quality scores or proportions of sections in each grade between the two staining methods. The proportions of sections that were classified as excellent or good were 96.7% and 98.3% for rapid and routine staining, respectively. CONCLUSIONS: In frozen breast tissue sections, rapid H&E staining may provide staining quality that is comparable to that of routine staining, while markedly reducing the staining time.

Novel Chromogens for Immunohistochemistry in Spatial Biology.

Spatial relations between tumor cells and host-infiltrating cells are increasingly important in both basic science and clinical research. In this study, we have tested the feasibility of using standard methods of immunohistochemistry (IHC) in a multiplex staining system using a newly developed set of chromogenic substrates for the peroxidase and alkaline phosphatase enzymes. Using this approach, we have developed a set of chromogens characterized by (1) providing fine cellular detail, (2) non-overlapping spectral profiles, (3) an absence of interactions between chromogens, (4) stability when stored, and (5) compatibility with current standard immunohistochemistry practices. When viewed microscopically under brightfield illumination, the chromogens yielded the following colors: red, black, blue, yellow, brown, and green. By selecting compatible color combinations, we have shown feasibility for four-color multiplex staining. Depending on the particular type of analysis being performed, visual analysis, without the aid of computer-assisted image analysis, was sufficient to differentiate up to four different markers.

Assessing Different Histological Preparations for Reconstruction of Astrocyte Tridimensional Structure.

Astrocytes are ubiquitous in the brain and spinal cord and display a complex morphology important for the local interactions with neighboring cells, resulting in the modulation of circuit function. Thus, studies focusing on astrocyte physiology in the healthy and diseased brain generally present analyses of astrocytic structure. The labeling method used to visualize the astrocytic structure defines the morphological level to observe and may vary depending on the anatomical sub-regions. The method choice may significantly affect our understanding of their structural diversity. The main goal of this work was to identify a straightforward and efficient protocol for labeling and reconstructing a detailed astrocytic structure to apply and validate in different brain tissue preparations across laboratories. For that, we explored different tissue processing protocols before GFAP labeling to determine the most effective method for reconstructing astrocytic backbones in the mouse hippocampus. Our results show that the reconstruction of astrocytic structure in vibratome sections labeled by free-floating immunofluorescence protocol provides a more practical method to achieve a higher level of detail and arbor complexity in astrocyte backbone reconstruction. Free-floating immunofluorescence labeling is the most reliable method for obtaining better antibody penetration and more detailed astrocyte structure. Finally, we also show that introducing an antigen retrieval step appears useful for visualizing more complete structural details.

Improvement of gram staining effect by ethanol pretreatment and quantization of staining image by unsupervised machine learning.

In this study, we propose an Ethanol Pretreatment Gram staining method that significantly enhances the color contrast of the stain, thereby improving the accuracy of judgement, and demonstrated the effectiveness of the modification by eliminating unaided-eye observational errors with unsupervised machine learning image analysis. By comparing the traditional Gram staining method with the improved method on various bacterial samples, results showed that the improved method offers distinct color contrast. Using multimodal assessment strategies, including unaided-eye observation, manual image segmentation, and advanced unsupervised machine learning automatic image segmentation, the practicality of ethanol pretreatment on Gram staining was comprehensively validated. In our quantitative analysis, the application of the CIEDE2000, and CMC color difference standards confirmed the significant effect of the method in enhancing the discrimination of Gram staining.This study not only improved the efficacy of Gram staining, but also provided a more accurate and standardized strategy for analyzing Gram staining results, which might provide an useful analytical tool in microbiological diagnostics.

Immunofluorescence staining of chloroplast proteins with frozen sections of plant tissues.

KEY MESSAGE: Immunofluorescence staining with frozen sections of plant tissues and a nest tube is convenient and effective, and broadens the applicability of immunofluorescence staining. Immunofluorescence staining is an indispensable and extensively employed technique for determining the subcellular localization of chloroplast division proteins. At present, it is difficult to effectively observe the localization of target proteins in leaves that are hard, or very thin, or have epidermal hair or glands with the current immunofluorescence staining methods. Moreover, signals of target proteins were predominantly detected in mesophyll cells, not the cells of other types. Thus, the method of immunofluorescence staining was further explored for improvement in this study. The plant tissue was embedded with 50% PEG4000 at -60℃, which was then cut into sections by a cryomacrotome. The sections were immediately immersed in fixation solution. Then, the sample was transferred into a special nested plastic tube, which facilitated the fixation and immunofluorescence staining procedures. The use of frozen sections in this method enabled a short processing time and reduced material requirements. By optimizing the thickness of the sections, a large proportion of the cells could be well stained. With this method, we observed the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis and various chloroplast division mutants. Meanwhile, the localization of FtsZ1 was also observed not only in mesophyll cells, but also in guard cells and epidermal cells in a lot of other plant species, including many species with hard leaf tissues. This method is not only easy to use, but also expands the scope of applicability for immunofluorescence staining.

[Optimal melanin removal methods for HE staining, immunohistochemistry and molecular detection].

Objective: To seek the optimal melanin-removal method for hematoxylin and eosin (HE) staining, immunohistochemistry and molecular detection. Methods: Thirty-eight paraffin tissue samples of malignant melanoma diagnosed at the Fujian Cancer Hospital, Fuzhou, China between January 2018 and March 2022 were collected and used to make a tissue microarray. Melanin in these cases was removed using warm hydrogen peroxide, double oxidation depigmentation, modified potassium permanganate-oxalic acid or trichloroisocyanuric acid, followed by HE staining. The cases were divided into two cohorts: one was subject to the one of the above four methods to remove melanin first, followed by immunohistochemistry (SOX-10, Ki-67, HMB45 and Melan A), while the other was subject to immunohistochemical staining first and then a melanin removal. Following that, seventeen melanin-rich paraffin tissue samples were collected and depigmented using the methods described above. DNA extraction was then done, followed by assessments of DNA content and quality. Moreover, the completeness of melanin removal, the effect on HE and immunohistochemical staining, and the quality of DNA were compared between the depigmented methods. Results: Regarding the effectiveness of melanin removal, the modified potassium permanganate-oxalic acid and the warm hydrogen peroxide methods were the most effective, and both showed residual melanin in only 5.26% (2/38) of the cases. The trichloroisocyanuric acid method showed residual melanin in 10.53% (4/38) of the cases. The worst was the double oxidation depigmentation method, which showed pigment residue in 15.79% (6/38) of the cases. For HE staining, the percentage of good staining with the warm hydrogen peroxide method was 92.11%, higher than the other three methods. For immunohistochemical staining, the mean staining scores of immunohistochemistry first followed by melanin removal with modified potassium permanganate-oxalic acid, double oxidation and trichloroisocyanuric acid were 20.84, 26.63 and 35.02, respectively. These immunohistochemical staining scores were higher than those of melanin removal first followed by immunohistochemistry (8.70, 15.41 and 21.22, respectively). The mean staining score of melanin removal by warm hydrogen peroxide method followed by immunohistochemistry was 33.57, superior to that of immunohistochemistry followed by the melanin removal (19.96). Moreover, the staining scores of HMB45, MelanA and Ki-67 with immunohistochemical staining followed by trichloroisocyanuric acid method were 36.45, 33.79, and 36.24, respectively, while the staining score of SOX10 with melanin removal by warm hydrogen peroxide followed by immunohistochemistry was 34.39. The DNA was significantly degraded by modified potassium permanganate-oxalic acid, double oxidation depigmentation and trichloroisocyanuric acid, whereas the mean concentration of DNA extracted after melanin removal by hydrogen peroxide method was 59.59 µg/L, substantially higher than that of DNA extracted without melanin removal (30.3 µg/L, P=0.001). The A260/A280 of DNA extracted after melanin removal by hydrogen peroxide was between 1.8 and 2.0 in all cases, and the A260/A230 was above 2.0 in sixteen cases, suggesting high purity of DNA. However, the DNA extracted without removing the melanin showed poor purity, with A260/A280 below 1.8 in eight cases and A260/A230 below 2.0 in sixteen cases. Conclusions: Warm hydrogen peroxide showed the least melanin residue, superior HE staining and a minimal effect on DNA purity/quality compared to the other three methods. It thus appears most suitable for PCR, NGS and other molecular detection. Melanin removal with trichloroisocyanuric acid after immunohistochemical staining has the least melanin residual, and thus could be the most convenient and efficient. However, it is noted that the efficacy of the same depigmentation method varies with different antibodies. Therefore, the optimal depigmentation method should be selected based on the specific markers of interest.

TSWIFT, a novel method for iterative staining of embedded and mounted human brain sections.

Comprehensive characterization of protein networks in mounted brain tissue represents a major challenge in brain and neurodegenerative disease research. In this study, we develop a simple staining method, called TSWIFT, to iteratively stain pre-mounted formalin fixed, paraffin embedded (FFPE) brain sections, thus enabling high-dimensional sample phenotyping. We show that TSWIFT conserves tissue architecture and allows for relabeling a single mounted FFPE sample more than 10 times, even after prolonged storage at 4 °C. Our results establish TSWIFT as an efficient method to obtain integrated high-dimensional knowledge of cellular proteomes by analyzing mounted FFPE human brain tissue.