Cell-type-specific Labeling of Endogenous Proteins Using the Split GFP System in .

i. Accurate identification of the locations of endogenous proteins is crucial for understanding their functions in tissues and cells. However, achieving precise cell-type-specific labeling of proteins has been challenging . A notable solution to this challenge is the self-complementing split green fluorescent protein (GFP ) system. In this paper, we present a detailed protocol for labeling endogenous proteins in a cell-type-specific manner using the GFP system in fruit flies. This approach depends on the reconstitution of the GFP and GFP fragments, creating a fluorescence signal. We insert the fragment into a specific genomic locus while expressing its counterpart, , through an available Gal4 driver line. The unique aspect of this system is that neither GFP nor GFP alone emits fluorescence, enabling the precise detection of protein localization only in Gal4-positive cells expressing the GFP tagged endogenous protein. We illustrate this technique using the adhesion molecule gene ( ) as a model, highlighting the generation and validation of GFP protein trap lines via Minos-mediated integration cassette (MiMIC) insertion. Furthermore, we demonstrate the cell-type-specific labeling of Ten-m proteins in the larval brains of fruit flies. This method significantly enhances our ability to image endogenous protein localization patterns in a cell-type-specific manner and is adaptable to various model organisms beyond fruit flies.