Lipid droplets have emerged as dynamic organelles involved in diverse cellular processes beyond simple lipid storage. In plants and cyanobacteria, growing evidence highlights their importance in stress adaptation and signaling, yet methods to study their structure and purity remain limited. Traditionally, in situ transmission electron microscopy (TEM) has been used to visualize lipid droplets within intact cells. While powerful, this approach cannot easily evaluate isolated lipid droplets or confirm their purity. In this protocol, we describe a rapid method for preparing and visualizing cyanoglobule lipid droplets isolated from cyanobacteria. The isolated droplets are directly processed for TEM using negative staining with uranyl acetate, providing a straightforward and efficient workflow. The procedure can be applied broadly to lipid droplets from diverse organisms, independent of species or cellular origin. This protocol offers a simple, fast, and widely applicable approach to assessing lipid droplets, expanding the toolkit for researchers studying their structure and function.Key features• Provides a brief and detailed method to visualize cyanoglobule lipid droplets.• Offers a rapid workflow (~1–2 h from preparation to imaging), enabling efficient sample processing and high-throughput analysis.• Employs negative staining and TEM to directly assess droplet morphology and purity without complex sample preparation.• Applicable to isolated lipid droplets from diverse organisms, making it a broadly useful tool beyond cyanobacteria.

Adipose tissue macrophages (ATMs) critically influence obesity-induced inflammation and metabolic dysfunction. Recent studies identified distinct ATM subsets characterized by markers such as CD11c, CD9, and Trem2, associated with pro-inflammatory and metabolically activated states. This protocol outlines a detailed, reproducible methodology for isolating, characterizing, and sorting these ATM subsets from murine epididymal white adipose tissue (eWAT) using multicolor flow cytometry. Key steps include stromal vascular fraction (SVF) isolation, immunophenotyping, sequential gating strategies, and fluorescence-activated cell sorting (FACS) for downstream gene expression analysis. The protocol was validated in diet-induced obese (DIO) mice treated with the IRE1 RNase inhibitor STF-083010, demonstrating its utility for studying ATMs in the context of obesity and metabolic disease.Key features• Detailed isolation and identification of multiple ATM subsets from eWAT.• Compatible with comprehensive flow cytometric analyses and fluorescence-activated cell sorting (FACS).• Facilitates downstream gene expression profiling from sorted ATM subsets.• Validated using a metabolic intervention (IRE1 RNase inhibitor STF-083010) in a mouse obesity model.

Circular RNAs (circRNAs) are covalently closed RNA molecules known for their increased stability compared to linear RNAs. Synthetic circRNAs are being developed as RNA therapeutics, while natural circRNAs are being investigated for their biological roles in eukaryotes and their potential as disease biomarkers. Consequently, the accurate detection and validation of circRNAs is crucial for advancements in both fundamental RNA research and biotechnological applications. Common methods for circRNA validation involve RT-PCR using divergent primers, followed by sequencing across the circRNA junction. However, most described methods are high-throughput approaches that require time-consuming RNA processing steps, and they are unable to detect highly structured circRNAs. Additionally, methods for low-throughput sequencing of small circRNAs (<150 nt) require cloning prior to sequencing. A simplified protocol for the validation of circRNA sequences irrespective of structure, sequence complexity, and length has not yet been described. In this method, we describe an improved RT-PCR protocol for circRNA detection by using UltraMarathonRT® (uMRT), a highly processive reverse transcriptase. Unlike other reverse transcriptases, uMRT can reverse-transcribe large, structured circRNAs of varying sizes, at ambient temperatures, enabling sequencing of the resulting concatemeric amplicons generated by RT-PCR and other methods. Using this method, we sequenced circRNAs containing highly structured internal ribosome entry sites commonly utilized in synthetic circRNAs, natural circRNAs containing repetitive elements, and small circRNAs, all without the need for cloning. With this new platform, we offer a protocol for the precise detection of nearly any circRNA species.Key features• This protocol shortens current methods for circRNA detection and sequencing by sequencing RT-PCR products directly, without the need for cloning or processing the PCR product.• This protocol describes a simple and cost-effective RT-PCR method for single circRNAs.• The highly processive UltraMarathonRT (uMRT) functions at ambient conditions, reducing circRNA degradation.• This protocol enables sequencing of circRNAs that are below 150 nt as well as larger circRNAs.• This protocol enables the detection of structured and very large circRNAs.

Synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are 21-nucleotide small RNAs designed to induce highly specific and efficient gene silencing in plants. Traditional approaches rely on the transgenic expression of ~1 kb TAS precursors, which limits their use in non-model species, under strict GMO regulations, and in size-constrained expression or delivery systems. This protocol describes a rapid workflow for the design, assembly, and delivery of syn-tasiRNAs derived from much shorter precursors, referred to as minimal precursors. The pipeline includes in silico design of highly specific syn-tasiRNA sequences, cloning of minimal precursors into plant expression or potato virus X (PVX)-based viral vectors through Golden Gate or Gibson assembly, and delivery to plants through Agrobacterium-mediated expression or by spraying crude extracts containing recombinant PVX expressing the minimal precursors. These methodologies make syn-tasiRNA-based tools more accessible and broadly applicable for plant research and biotechnology across diverse species and experimental contexts.Key features• Syn-tasiRNAs allow the simultaneous silencing of multiple genes with high specificity, as they are computationally designed to avoid off-target effects.• This protocol describes the design and obtention of syn-tasiRNAs for the simultaneous silencing of one or several endogenous genes in any plant species.• This protocol also describes a non-transgenic alternative for applying syn-tasiRNAs to plants using a viral vector to induce whole-plant gene silencing.• This protocol can also be applied to induce antiviral protection against pathogenic viruses, reducing viral mutational escapes when expressing multiple syn-tasiRNAs targeting different viral sites.

Formalin-fixed paraffin-embedded (FFPE) slides are essential for histological and immunohistochemical analyses of organoids. Conventional preparation of FFPE slides from organoids embedded in basement membrane extract (BME) presents several challenges. During the fixation step, dehydration often causes collapse of the BME, which normally supports the three-dimensional architecture of organoids. As a result, organoids may lose their original morphology, particularly in the case of cystic or structurally delicate types, leading to distortion and reduced reliability in downstream histological evaluation. Here, we introduce a straightforward protocol that improves the reliability of FFPE slide preparation for BME-based organoids by enhancing sample integrity and sectioning quality. By using 2% agarose as a mold during the embedding process, organoids grown in BME were effectively stabilized, enabling reliable preservation of their morphology throughout FFPE slide preparation. This method effectively addresses the difficulties in processing structurally delicate organoids and allows robust preparation of diverse cancer organoid morphologies—such as cystic, dense, and grape-like structures—while maintaining their native three-dimensional architecture. Our approach simplified the technical process while ensuring reliable histopathological analysis, making it a valuable tool for cancer research and personalized medicine.Key features• FFPE preparation applicable to diverse cancer organoid morphologies, including cystic, dense, and grape-like, while preserving three-dimensional architecture.• Agarose molding allows intact retrieval and fixation of BME domes, preventing collapse and maintaining organoid 3D architecture during FFPE preparation.• Compatible with diagnostic IHC/IF markers (pan-CK, CK19, p63, Ki-67, p53) across cancer organoids.

Cryo-electron tomography (cryo-ET) is the main technique to image the structure of biological macromolecules inside their cellular environment. The samples for cryo-ET must be thinner than 200 nm, which is not compatible with micron-sized cells. A focused ion beam (FIB), in conjunction with a scanning electron microscope (SEM) to navigate the sample, can be used to ablate material from vitrified cells such that a thin lamella remains. However, the preparation of lamellae with a FIB-SEM is blind to the location of specific cellular structures and biomolecules. Furthermore, the thickness and uniformity of lamella, while crucial for high-quality tomograms, cannot be established accurately with the FIB-SEM. These limitations strongly affect the success rate for cryo-ET on FIB-milled lamellae and thereby the total throughput of the workflow. To mitigate these problems, a coincident light, electron, and ion beam cryo-microscope was developed by retrofitting a fluorescence microscope, cryogenic microcooler, and piezo stage on a FIB-SEM. The fluorescence of molecules of interest can be monitored in real time while milling to ensure the final lamella contains the structure of interest. In addition, reflected light microscopy can be used for thickness and quality control of the lamella. In this protocol, we will describe how the coincident microscope can be used to prepare lamellae from vitrified cells.Key features• Step-by-step protocol for fluorescence-guided FIB-milling with a coincident three-beam cryogenic microscope as described in [1].• Details about sample loading and unloading, as well as the lamella milling workflow with graphical explanations.• Quality control of lamella, including thickness, uniformity, and ice contamination.

Alto TM (Localized Surface Plasmon ResonanceLSPR) SPR (Digtal Microfluidic, DMF) Alto Fc (FcR) IIb ( CD32) IgG

In the intestinal epithelium, intraepithelial lymphocytes (IELs) coexist with intestinal epithelial cells (IECs). The IELs have an important role in defending the intestinal tract against pathogens and eliminating tumor cells. Anomalies in the absolute IEL count have been reported in various digestive diseases. IELs are typically counted using histologic techniques or under light microscopy after isolation of the epithelium. However, these techniques can introduce bias, which might account for the discrepancies in counts from one study to another. Here, we describe a flow cytometry assay for determining the absolute IEL count and the IEL/IEC ratio. We combined a conventional epithelial isolation method with a BD TruCountTM bead-based absolute counting technique to quantify IELs (CD45+ CD326/EpCAM- CD103+CD3+) and IECs (CD45- CD326/EpCAM+) in a C57BL/6 mouse model. Key features • Intraepithelial lymphocytes (IELs) play a crucial role in maintaining mucosal integrity and defending against pathogens. • Conventional manual counting of IELs using a hemocytometer relies heavily on the operator's expertise. • Flow cytometry offers a more standardized approach to cell counting. • Using TruCountTM beads to quantify IELs and intraepithelial cells (IECs) by flow cytometry and assess their ratio ensures reproducibility and comparison with immunohistochemical methods. Graphical Overview.

Research on brain disorders, particularly in the field of oncology, requires in vivo models to evaluate various therapeutic approaches, including intracerebral drug delivery. To meet this requirement, the implantation of intracerebral cannulas offers a reliable method for administering candidate therapeutics directly into the brain. This protocol describes a surgical technique for cannula implantation in mice, enabling repeated administration of therapeutic compounds in the context of glioblastoma treatment. The method was designed with an emphasis on using accessible, easy-to-handle, and sterilized tools to optimize surgical outcomes. Particular attention was also given to animal welfare, notably through refined procedures for asepsis, anesthesia, and postoperative care.Key features• This protocol requires specific equipment and surgical mice expertise.• This protocol is dedicated to brain disorders and cancer models, with a particular emphasis on locoregional delivery of drugs and derivatives.