Protocol for in vitro immunofluorescence staining in a Transwell co-culture system
Year: 2025
Type: article
Abstract: Immunofluorescence (IF) is a key technique for localizing target molecules. Here, we present a protocol for in vitro IF staining optimized for skeletal muscle myotube formation cultured on Transwell inserts. We describe steps for primary cell culture, fixing, permeabilization, and antibody incubation. We then detail procedures for quantifying myotube differentiation by IF microscopy to calculate fusion index. This protocol will enable direct IF staining on cell monolayer seeded on permeable support in co-culture or other Transwell studies. • Steps to capture myotubes cultured on permeable support using fluorescent microscopy • Procedures for primary cell culture, fixing, permeabilization, and antibody incubation • Instructions to quantify myotube differentiation to calculate fusion index with ImageJ Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Immunofluorescence (IF) is a key technique for localizing target molecules. Here, we present a protocol for in vitro IF staining optimized for skeletal muscle myotube formation cultured on Transwell inserts. We describe steps for primary cell culture, fixing, permeabilization, and antibody incubation. We then detail procedures for quantifying myotube differentiation by IF microscopy to calculate fusion index. This protocol will enable direct IF staining on cell monolayer seeded on permeable support in co-culture or other Transwell studies.
Source: STAR Protocols
Institutions: Eastern Health, Monash University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,550
Award: G6162039248
OpenAlex ID: W4415535952