Drosophila ovary is an excellent model for studying material transportation and dynamics. Here, we present a protocol for live imaging of transposon products and host proteins in Drosophila ovaries. We describe steps from ovary dissection, ovariole isolation, and mounting to live tracking of fluorescently labeled proteins with single-cell resolution. Furthermore, we detail procedures of image processing for jitter correction and kymograph analysis. This protocol can be applied to studies on the ...

Accurate prediction of tumor prognosis, understanding molecular heterogeneity, and identifying druggable targets are critical for advancing precision oncology. Here, we present a protocol to analyze aging-associated prognosis and molecular heterogeneity in gastric tumors using a transcriptome-based, machine learning-driven approach. We describe procedures for developing a prognostic model using robust gene signature, identifying molecular subtypes, and building a machine learning classifier for ...

Here, we detail a protocol to identify an antigen-specific B cell population using SpyCage reagents tailored to bind MSP1-19 antigen in a rodent malaria model. We describe steps for the preparation of splenic single-cell suspension, B cell enrichment, and staining to facilitate sorting of live cells for downstream applications such as single-cell RNA, V(D)J, and assay for transposase-accessible chromatin (ATAC) sequencing. For complete details on the use and execution of this protocol, please re...

Here, we present a protocol for CRISPR knockout screening in human induced pluripotent stem cell (hiPSC)-derived microglia (iMGL) using lentiviral delivery of CRISPR-Cas9 and co-transduction of VPX virus-like particles (VPX-VLPs). We first describe large-scale production of iMGL from hiPSCs, production of the lentiviral and VPX-VLP libraries, and titration. Next, we describe how to perform a pooled CRISPR screen for phagocytosis including the computational analysis pipeli...

Here, we present a protocol to quantify and analyze multiplexed fluorescence in situ hybridization (mFISH) data using two open-source tools, FijiFISH and RUHi. FijiFISH, an ImageJ-based plugin, enables image registration, cell segmentation, and gene expression quantification. RUHi, an R-based package, supports dimensionality reduction, clustering, and visualization through both code and a Shiny app. The protocol also accommodates experimentally induced exogenous fluorophores, providing multimoda...

STAR Protocols is an open access, peer-reviewed journal from Cell Press. We offer structured, transparent, accessible, and repeatable step-by-step experimental and computational protocols from all are...

The stiffness of extracellular matrices is critical for cellular functions such as growth and differentiation. Here, we present a protocol for culturing cells on stiffness-modulated collagen gels by adding genipin, an amine crosslinker with low cytotoxicity. We describe the steps for preparing collagen gels with genipin, culturing the cells, and immunofluorescent staining of the cells on the gels. This protocol has potential applications in the analysis of function or protein/gene expression in ...

Overweight and obesity are multifactorial health problems requiring innovative analytical approaches. We present a protocol to develop a population-based individualized intervention planning assistant (PIIPA) using supervised machine learning, Shapley additive explanations (SHAP), and a long short-term memory (LSTM) model. We describe steps for constructing interpretable machine learning (ML) models, identifying key predictors, and simulating BMI trajectories under interventions. We then detail ...

Patients diagnosed with cancer of unknown primary (CUP) have poor prognoses, highlighting the need to identify primary sites for targeted therapeutic intervention. Here, we present a protocol to predict the primary sites of CUP based on gene expression data using BPformer. We describe steps for installing software, collecting and preprocessing data, and training the BPformer model. We then detail procedures on using BPformer via a visualized web server. For complete details on the use and execut...

Here, we present a protocol for infecting Aedes aegypti mosquitoes with Zika virus (ZIKV) (Flaviviridae) using a glass artificial feeder that mimics natural feeding conditions. We describe steps for preparing adult mosquitoes, assembling the feeder, mixing the infectious blood meal, and conducting infection experiments. The feeder incorporates a bovine gut membrane and a thermostatic circulator to maintain the blood meal at 37°C, ensuring consistent feeding and reproducible infection outcomes. F...

Here, we present a protocol for preserving fluorescent markers using tissue clearing and immunolabeling in mouse tissue such as sciatic nerve, gut, and spinal cord. We describe steps for maintaining tetrahydrofuran (THF) under an inert atmosphere, EZ clearing, immunolabeling, imaging with custom lightsheet microscopy setups, and post-processing. This protocol was refined to ensure stable and reliable clearing across various tissue types. Key improvements include inert atmosphere THF handling, ti...

Here, we present a protocol for fabricating a drone-deployable electric current sensor for real-time, remote monitoring of electric currents in overhead transmission lines. We describe steps for designing and manufacturing the board in Autodesk EAGLE and the signal processing units. We then detail procedures for programming routines on the Arduino Due to process data and curve visualization environment for Bluetooth-received data in MATLAB. The system supports data exchange up to 60 meters, cont...

Formaldehyde is emerging as a key signaling modulator. Here, we present Trami, a protocol that traps endogenous formaldehyde in live cells and converts its transient signals into stable proximal protein labels for subsequent signaling study. We describe probe design, live-cell administration, and imaging procedures for signaling deconvolution. Trami offers a powerful tool for elucidating formaldehyde-mediated signaling pathways. For complete details on the use and execution of this protocol, ple...

Research on inducing beige adipocytes to combat obesity and its comorbidities is growing, with stromal vascular fraction (SVF) from white adipose tissue showing potential to differentiate into both white and beige adipocytes. Here, we present a protocol for isolating the SVF from the iWAT (inguinal white adipose tissue) of mice and differentiating them into mature beige adipocytes. This protocol can be used to analyze the translational regulation and metabolic processes of beige adipocytes in vi...

Chemostat growth chambers measure optical density over time and require manual calculation of growth rates. Here, we present chemostat_regression, R software that enables users to automatically identify chemostat cycles and estimate growth rate using a linear regression approach. We describe steps for creating requisite software environment(s), formatting input data, executing the software via command line/RStudio/R-Shiny, interpreting results, assessing the validity of results, and modifying in...

Proteins expressed in or translocated to the shoot apical meristem (SAM) often play pivotal roles in regulating plant development. Here, we present a step-by-step protocol for immunofluorescence imaging to analyze epitope (FLAG)-tagged proteins in the Arabidopsis SAM. This technique may be suitable to visualize mobile proteins because small epitope tags are less disruptive than a relatively large fluorescent protein fusion. For complete details on the use and execution of this protocol, please r...

Advances in sequencing technologies have enabled transcriptional profiling of previously understudied yet critical species, such as mosquitoes. Here, we present a protocol for multiplex whole-mount RNA fluorescence in situ hybridization combined with immunohistochemistry in the Anopheles gambiae brain. We describe steps for dissection, fixation, probe hybridization, hybridization chain reaction (HCR) probe detection, and primary antibody application. We then detail procedures for tissue preparat...

High-throughput CRISPR interference (CRISPRi) screens are invaluable for discovering novel functional genes, but applying such screens to long non-coding RNAs (lncRNAs) is more challenging. Here, we present a protocol for designing and executing pooled CRISPRi screens targeting lncRNAs using an abridged cell-type-specific dual single-guide RNA (sgRNA) library. We describe steps for library design and synthesis, followed by stable lentiviral transduction. We then provide guidelines for performing...

Targeted investigation of spatial transcriptomic data benefits from versatile integration of customized external information. Here, we present a protocol for embedding irregular field-of-view annotations and immunohistochemistry data into a Xenium dataset at the single-cell resolution using open-source tools. We detail steps for image registration and demonstrate how to integrate spatially aligned information into a Scanpy-compatible anndata object using a head and neck squamous cell carcinoma s...

Poly(ADP-ribose) polymerase 1 (PARP1) is a crucial DNA repair protein and a target of PARP inhibitors (PARPi), which suppress its catalytic activity while also altering its association with damaged chromatin. Here, we present a live-cell imaging protocol for obtaining high-quality kinetics of fluorescently labeled PARP1 and poly(ADP-ribose) (PAR)-binding proteins at micro-irradiation-induced DNA damage sites in untreated and PARPi-treated cells. This approach can be easily adapted to other DNA r...

Designer receptors exclusively activated by designer drugs (DREADDs) are a highly precise and effective method for manipulating neurons and other excitable cells. Here, we present an approach that combines viral anterograde transsynaptic transport with Gαi/o DREADD neuronal inhibition to control targeted neurons in a rat model. We describe how to implement this technique, from surgical the procedure to the behavioral assay. This protocol enables researchers to manipulate target neurons receiving...

CellMiner Cross-Database (CellMinerCDB) is an interactive web application for integrating and analyzing molecular and pharmacological data across human cancer cell lines. Here, we detail the setup process, including installing necessary software, preparing compatible datasets, and customizing configuration files; we use sarcoma data as an example. The protocol involves data loading, software configuration, and deployment to enable univariate and multivariate analyses. For complete details on the...

Microfluidics has versatile applications in the field of biosensors. We present a protocol for assembling a microfluidic system to rapidly measure cortisol in saliva. This protocol prepares microfibrous reactors immobilized with antibodies as an alternative to a 96-well plate and integrates a flow system, reactor platform, and detection device. The output current signal directly reflects cortisol levels for rapid evaluation. For complete details on the use and execution of this protocol, please ...