STAR Protocols最新文献

Here, we present a protocol to express anti-viral nanobodies or antigenic proteins anchored on the bacterial cell surface through the engineering of probiotic E. coli Nissle 1917 (EcN). We describe steps for bacterial transformation, protein validation, outer membrane vesicle (OMV) isolation, and in vivo administration. We then detail procedures for immunological assessment through ELISA, neutralization assay, and flow cytometry analysis. This protocol can be adapted for different therapeutic targets beyond SARS-CoV-2. For complete details on the use and execution of this protocol, please refer to Kamble et al.¹.

The common marmoset (Callithrix jacchus) is an important nonhuman primate model in biomedical research, but the lack of a standardized necropsy protocol limits reproducibility and data quality. Here, we present a protocol for necropsy and tissue processing of the common marmoset (Callithrix jacchus). We describe steps for anesthesia, external observation, laparotomy, blood collection, organ sampling, and fixation. We then detail procedures for trimming, tissue processing, and embedding. This protocol enables researchers to obtain high-quality, reproducible samples for histopathological analyses.

Identification of interacting proteins, particularly those mediating protein degradation, remains technically challenging. The transcription factor BZR1 in Arabidopsis is activated by brassinosteroid (BR) signaling to promote plant growth and is degraded under starvation conditions to restrict growth and promote survival. Here, we present a protocol for identifying BZR1 interactors in plants under sugar starvation using ¹⁵N stable isotope labeling (SIL) followed by immunoprecipitation and quantitative mass spectrometry (IP-MS) analysis. This protocol has broad potential applications in studying the protein interactome. For complete details on the use and execution of this protocol, please refer to Zhang et al.¹.

In vivo optogenetic stimulation is a powerful approach for dissecting neuronal circuit function, yet it can inadvertently lead to activation of endogenous retinal opsins, producing off-target responses that propagate to the cortex and therefore confound experimental interpretation. Here, we present a protocol for optical device implantation in the mouse brain that minimizes light leakage from the implant and skull. We also describe in vivo electrophysiological techniques to assay off-target neuronal activity and ensure its amelioration. For complete details on the use and execution of this protocol, please refer to Weiler et al.¹.

Adult hippocampal neurogenesis (AHN) results in the generation of new neurons throughout adulthood, thereby contributing to key hippocampal functions such as learning and mood regulation. Here, we present a protocol for the immunofluorescence-based detection of AHN markers in fresh-frozen human hippocampal sections. We describe steps for tissue fixation, antibody incubation, and autofluorescence elimination. We then detail procedures for image acquisition and quantification. This protocol allows the reliable identification of distinct cell populations and could be adapted for broader immunohistochemical applications. For complete details on the use and execution of this protocol, please refer to Márquez-Valadez et al.¹.

Modeling neurological disorders is challenging due to differing functional genomics and phenotypes among species. Here, we present a protocol for morphogen-guided differentiation of human induced pluripotent stem cells (hiPSCs) into microglia, astrocytes, and mixed cortical cultures (MCCs) for studying human brain disorders. We describe steps for enhancing microglial production using hypoxia and implementing quality-control measures for astrocyte and MCC differentiations. We detail knockout serum replacement procedures for serum-free astrocytes. This protocol enables cell-type-specific investigation of disease mechanisms and drug screening.

Here, we present a protocol for generating postmortem human brain tissue samples suitable for volume electron microscopy (vEM) imaging via focused ion beam-scanning electron microscopy (FIB-SEM). We describe steps for immersion fixation, sectioning, long-term cryopreservation, and enhanced-contrast staining that produce samples suitable for high-resolution FIB-SEM. This approach generates ultrastructure of sufficiently high preservation to enable both dense and targeted three-dimensional (3D) reconstructions of cellular, subcellular, synaptic, and organellar features in postmortem human brain samples. For complete details on the use and execution of this protocol, please refer to Glausier et al.¹.

Dual spatial transcriptomics analysis offers profiling of host- and pathogen-specific transcriptional patterns in infected tissues to establish pathoadaptation signatures and predict infection outcomes. Here, we present a protocol for tissue processing, imaging, selection of regions of interest, library preparation, sequencing, and analysis pipeline. This protocol has a potential application for the analysis of any infected tissue. For complete details on the use and execution of this protocol, please refer to Zhou et al.¹.

Available mouse models for pancreatic ductal adenocarcinoma (PDAC) are limited by slow tumor development and failure to recapitulate key stromal and immune characteristics. Here, we present a protocol for generating a collagen hydrogel mouse model for orthotopic PDAC. We describe steps for embedding mouse pancreatic cancer cells in a dense collagen hydrogel and surgically implanting it into the mouse pancreas. Mouse PDAC tumors typically reach 1 cm in diameter by 10 days after implantation and show immune and stromal cell recruitment. For complete details on the use and execution of this protocol, please refer to Korah et al.¹.

CD70 has emerged as a promising immunotherapeutic target in renal cell carcinoma (RCC), with both CD70-directed monoclonal antibodies and chimeric antigen receptor (CAR)-based therapies currently under development. Here, we describe a protocol for the generation of human CD70-directed allogeneic CAR-natural killer T (NKT) (^AlloCAR70-NKT) cells derived from cord blood CD34⁺ hematopoietic stem and progenitor cells (HSPCs) using a clinically guided culture. Furthermore, we describe the therapeutic efficacy of ^AlloCAR70-NKT cells in mediating cytotoxic activity against RCC cell lines in vitro. For complete details on the use and execution of this protocol, please refer to Li et al.¹.