Super-resolution has allowed us to image areas previously inaccessible due to the limits of standard microscopy techniques. Here, we present a protocol to determine the astroglial coverage of synapses. We detail the steps to obtain and label mouse brain slices followed by imaging using 3D stimulated emission depletion (STED) microscopy and analysis using a custom-made Fiji plug-in, with imaging at a lateral resolution of 80 nm. This technique ascertains the interaction between astrocytes and the glial cell’s role in signaling and brain region function.
The need for compact tubular ice machines is paramount to reducing investment costs, installation space, and heat loss. Here, we present a protocol for designing and fabricating a monolithic integrated freezer structure for tubular ice machines, thereby consolidating the freezing chamber, heat exchanger, accumulator, and water distribution tank. We detail the steps for setting up the system and analyzing the coefficient of performance (COP). This protocol has the potential for direct application in next-generation industrial ice-making systems to optimize space.For complete details on the use and execution of this protocol, please refer to Nguyen et al.1,2
Barcoded Saccharomyces cerevisiae strains enable pooled genetic analyses across many genotypes. Here, we describe a protocol for generating stable barcoded haploid strains from natural diploid isolates. The workflow includes high-throughput transformation, selection and validation of transformants, followed by sporulation and recovery of stable haploids. We also describe phenotypic and molecular validation methods. An optional en masse mating procedure generates pooled diploid offspring.For complete details on the use and execution of this protocol, please refer to Strauss et al.1
NF-κB transcription factor (TF) activation is heterogeneous across identically stimulated cells, shaping innate immune responses. Here, we present a protocol to quantify this heterogeneity in live Drosophila melanogaster S2∗ cells using dual reporters of NF-κB (Relish) subcellular localization and downstream transcription. We describe steps for reporter cell line generation, immune stimulation and time-lapse confocal imaging, and machine-learning-based single-cell analysis to classify signaling behaviors and predict transcriptional responses.For complete details on the use and execution of this protocol, please refer to Nawar et al.1
The intestine is home to more immune cells than any other non-lymphatic peripheral tissue, but research of these cells is stymied by the technical difficulties involved in isolating them. Here, we describe a protocol to isolate highly viable immune cells from murine intestinal epithelium and lamina propria. This protocol can be used to characterize cells by flow cytometry or single-cell transcriptomics, as in Crotts et al., or by other analysis methods.
