Q&A Feb 18, 2026

Group-based Minimum Spanning Tree Correlation NetworkAuthors: Camilo Espinosa Bernal, Nima Aghaeepour The R script outlined below and attached as a supplementary txt file represent a user-friendly template to generate a group-based MST of a correlation network of clinical features for which predictive multiomic models have been built. # "# Generic Multi-Omic MST (Minimum Spanning Tree) Template# ========================================================# INPUTS REQUIRED# ---------------# 1. cl...

Q&A Feb 10, 2026

Anti-acetylated tubulin IF – Nerve IFKaylee Wells April 2020  Day 1 Primary Antibody Solution:Anti-acetylated tubulin, T7451 mouse monoclonal (Located in small -20 ℃)1:1500 dilution in 0.5% BSA (BSA made in PBST) PBS x2 (10 min)Pap pen around the sections and let dry ( >1 hour)PBS (10 min)0.5% Trition X in PBST (5 min)Block in 0.5% BSA in PBST (>2 hours)Incubate with primary antibody (overnight) at 4 ℃ in humidification chamber Day 2 Secondary Antibody Solution:Goat-anti-mouse 488A11001 – ...

Q&A Feb 5, 2026

Preparation of acute brain slicesComplete growth media composition (with 5% serum and NGF+/GDNF+):Prepare 100ml slice culture with 5% serum Mix 40 ml MEM (Minimal essential medium, INV-42360032, Invitrogen), 20 ml BME (Basal medium eagle, INV-21010046, Invitrogen), 5 ml of 5% heat inactivated horse serum (INV-26050070, Invitrogen), 0.8 ml Glutamax (Thermo Fischer Scientific, Cat# 35050061), 0.8 ml Penstrep (Thermo Fischer Scientific, Cat# 15140122), 1.1 ml Glucose (45% w/v, Sigma-Aldrich, Cat# A...

Q&A Feb 5, 2026

Determination of BV2 Microglial Phagocytic Activity (Fluorescent Microspheres / FluoSpheres Assay; Flow Cytometry/Microscopy) OverviewThis assay quantifies phagocytosis of fluorescent carboxylate-modified microspheres by BV2 microglia. After exposure to the test compound, cells are incubated with FluoSpheres, washed to remove non-internalized beads, detached with Accutase containing Hoechst 33342 to identify nuclei, and analyzed by flow cytometry. Phagocytosis is reported as the percentage of Fl...

Q&A Jan 31, 2026

Lentiviral Packaging, Concentration, and Transduction  Introduction: This protocol describes the production, concentration, and usage of second generation VSV-G pseudo typed lentiviruses for gene delivery in mammalian tissue culture. Growing Packaging Cells: Grow 293T viral packaging cells in DMEM containing 4.5 g/L glucose and 110 mg/mL sodium pyruvate, supplemented with 10% FCS, 1% L-glutamine, and 1% penicillin/streptomycin. Passage 90% confluent cultures by trypsinization and re-seed 1:5 or ...

Q&A Jan 28, 2026

Immunofluorescence ProtocolQuintana lab Rinse sections of antifreeze solution with PBS pH 7.4........................…………………………................. 3x5’Block sections with PBS 0.2% Triton X-100 + 10% normal donkey serum (NDS) (9ml +1ml) ......…....... 1h at RTIncubate with primary antibody in PBS + 0.2% Triton X-100 + 1% NDS (9.9ml +0.1ml)...........…............ ON at 4ºCWash with PBS 0.2 Triton X-100.....................…………………………………………………….................. 3x5’Incubate with secondary antibody P...

Q&A Jan 26, 2026

Co-immunoprecipitation (co-IP) Following SUMOylation/Ubiquitination Perturbation(TAK-981/TAK-243 treatment with NEM-preserved, non-denaturing lysis)OverviewThis protocol describes the workflow for treating cells with TAK-243 (ubiquitin E1/UBA1 inhibitor) and/or TAK-981 (SUMO E1 inhibitor), harvesting cells under cold conditions, preparing NEM-preserved non-denaturing lysates, and performing co-immunoprecipitation (co-IP) for downstream immunoblot analysis. Because SUMO conjugates can be lost dur...

Q&A Jan 23, 2026

Bulk RNA-sequencing Differential Gene Expression Generation: With respect to bulk RNA-sequencing analysis, differentially expressed genes with respect to condition (pregnant/pseudopregnant/superovulation) and time (0.5 days-post-coitus (dpc) pregnant vs 1.5 dpc pregnant) were generated utilizing the Biojupies web-interface pipeline (https://maayanlab.cloud/biojupies/). Excel sheets containing differentially expressed genes were exported from Biojupies, and further manipulated with respect to Log...

Q&A Jan 14, 2026

Protocol for GSEA/GOTake the mean of forward and reverse experiments.Rank the list and submit to GSEA (Gene set Enrichment Analysis) against Gene-Ontology (GO) database. This software is free to download online and easy to use.Since we pre-ranked list use ‘Run GSEAPreranked’. Requires a list in .rnk format. This reflects the degree to which a gene set is overrepresented at the top or bottom of a ranked list of genesControl is a permutated dataset (1000 iterations)40 GO terms passed the threshold...

Q&A Jan 9, 2026

Setting up Drosophila primary cultures from RasV12-expressing genotypesThis is a detailed protocol for establishing primary cultures that has more details than the original method in a paper describing derviation of continuous lines from Drosophila culures (https://elifesciences.org/articles/85814#s4). Author for enquiries: Amanda Simcox, Department of Molecular Genetics, Ohio State University, Columbus OH 43210. simcox.1@osu.edu Materials and reagents:Egg laying medium: Separately autoclave sol...