STAR Protocols最新文献

Direct stochastic optical reconstruction microscopy (dSTORM) unveils ionotropic N-methyl-D-aspartate receptor (NMDAR) organization into discrete nanometer-size domains (nanoclusters) within the postsynaptic density (PSD) of glutamatergic synapses, tuning receptor signaling. Here, we present a protocol to perform 3D-dSTORM imaging of the NMDAR in organotypic and acute rat hippocampal brain slices by combining conventional dSTORM with fluorescent self-interference. We describe steps for sample preparation, immunohistochemistry, 3D-dSTORM acquisition, and image analysis to successfully super-resolve NMDAR nanodomains in three dimensions. For complete details on the use and execution of this protocol, please refer to Kellermayer et al.,¹ Bon et al.,² and Ferreira et al.³.

Aberrant mitochondrial function can lead to severe human diseases, including neurodegenerative diseases and cancer. Here, we describe a cell-based protocol for measuring different mitochondrial respiratory parameters using the high-resolution real-time Resipher system. We optimized this protocol on brain tumor stem cells cultured in three-dimensional spheroids. We provide essential optimization steps for cell seeding density, mitochondrial respiration modulator concentrations, running the assay, and data analysis. For complete details on the use and execution of this protocol, please refer to Burban et al.¹.

To investigate the role of hepatocyte genes in fasting metabolism, it is essential to analyze their expression across the entire 24-h circadian cycle. Here, we present a protocol to evaluate fasting metabolism and its relationship to the core circadian clock in mice. We describe steps for time-course fasting/feeding experiment setup and performing staggered fasting and refeeding for 24 h. We then detail procedures for evaluating oxidative substrate selection and rescuing defective oxidative metabolism through FGF21 and CPI-613 injections. For complete details on the use and execution of this protocol, please refer to Sun et al.¹.

The tri-methylation of histone H3 on K4 (H3K4me3) is a key epigenetic modification that is predominantly found at active gene promoters and is deposited by the complex of proteins associated with SET1 (COMPASS). CXXC zinc finger protein 1 (Cfp1) regulates this process by recruiting SET1 to chromatin and recognizing H3K4me3 via its plant homeodomain (Cfp1^PHD). Here, we present a protocol for the purification and crystallization of the Drosophila melanogaster Cfp1^PHD domain in complex with an H3K4me3 peptide (PDB: 9C0O). We describe steps for obtaining highly pure Cfp1^PHD and diffraction-quality crystals. We then detail procedures for rapidly identifying crystals containing the H3K4me3-bound form of the Cfp1^PHD domain. For complete details on the use and execution of this protocol, please refer to Grégoire et al.¹.

G-quadruplex and intercalated motif are quadruplex structures of which sequences are enriched in promoters. Here, we present a protocol for circular dichroism spectral analysis of the thermal stability of CpG-methylated quadruplex structures. We describe steps for preparing the oligonucleotide sample, measuring the circular dichroism spectrum of methylated quadruplex structures, and calculating thermodynamic parameters using Python 3. For complete details on the use and execution of this protocol, please refer to Kimura et al.¹.

Here, we present a protocol to decode Mandarin sentences from invasive neural recordings using a brain-to-text framework. We describe steps for preparing materials, including designing the sentence corpus and setting up electrocorticography (ECoG) recording systems. We then detail procedures for decoding, such as data preprocessing, selection of speech-responsive electrodes, speech detection, syllable and tone decoding, and language modeling. We also outline performance evaluation metrics. For complete details on the use and execution of this protocol, please refer to Zhang et al.¹.

Revealing the origins of alveolar epithelial stem cells is crucial for preventing and treating lung diseases. Here, we present a protocol for tracing alveolar type I (AT1) cells from mice in vivo using two distinct dual recombinase-mediated systems. We describe steps for model establishment, harvesting tissue, cryosection, immunofluorescence staining, and confocal imaging. We then detail procedures for analysis of image files and quantification. This protocol provides a foundation for elucidating the plasticity of AT1 cell fate during homeostasis and diseases. For complete details on the use and execution of this protocol, please refer to Liu et al.¹.

Identifying genes important for fitness in Candida albicans advances our understanding of this important pathogen of humans. Here, we present a functional genomics approach for assessing fitness through the quantification of strain-specific barcodes. We describe steps for library preparation, propagation of strains, genomic DNA extraction, amplification of barcodes, and sequencing. We then detail the computational analysis of data to determine effect size and statistical significance. For complete details on the use and execution of this protocol, please refer to Xiong et al.¹.

Monitoring intrapulmonary vasodilation in animal models is vital for understanding pulmonary vascular physiology and disease mechanisms. Here, we present a protocol for the real-time visualization and quantification of pulmonary microvascular dynamics in living mice using contrast echocardiography alongside microbubble injection. We describe steps for mouse preparation, baseline echocardiography, microbubble administration, and imaging to capture dynamic changes in pulmonary vasculature. Closely resembling clinical technique used in humans, this methodology provides an effective tool for investigating the pathophysiology of diverse cardio-pulmonary vascular diseases. For complete details on the use and execution of this protocol, please refer to Dandavate et al.¹.