Abstract
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 in vivo . A notable solution to this challenge is the self-complementing split green fluorescent protein (GFP (1-10/11) ) system. In this paper, we present a detailed protocol for labeling endogenous proteins in a cell-type-specific manner using the GFP (1-10/11) system in fruit flies. This approach depends on the reconstitution of the GFP (1-10) and GFP (11) fragments, creating a fluorescence signal. We insert the GFP (11) fragment into a specific genomic locus while expressing its counterpart, GFP (1-10) , through an available Gal4 driver line. The unique aspect of this system is that neither GFP (1-10) nor GFP (11) alone emits fluorescence, enabling the precise detection of protein localization only in Gal4-positive cells expressing the GFP (11) tagged endogenous protein. We illustrate this technique using the adhesion molecule gene teneurin-m ( Ten-m ) as a model, highlighting the generation and validation of GFP (11) 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.