Protocol for in vitro immunofluorescence staining in a Transwell co-culture system

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.