High-Resolution Imaging of Intracellular Trafficking of B Cell Receptor Using Specific Hybridization Internalization Probe (SHIP)

: Hernández-Pérez, Sara, Mattila, Pieta K.

: Palash Chandra Maity

Publisher: Humana Press Inc.

: 2025

: B-Cell Receptor Signaling - Methods and Protocols

: Methods in Molecular Biology

: 2909

: 73

: 82

: 978-1-0716-4441-6

: 978-1-0716-4442-3

: 1064-3745

: 1940-6029

DOI: https://doi.org/10.1007/978-1-0716-4442-3_6

: https://link.springer.com/protocol/10.1007/978-1-0716-4442-3_6

Recent advancements in microscopy have greatly expanded our understanding of intracellular traffic. Yet, due to the inherent characteristics of B cells, such as their small size and high receptor density on the plasma membrane, visualization of internalized cargo or receptors remains challenging. This challenge is particularly pronounced in the case of the B cell receptor (BCR), where accurate detection of internalized, antigen-bound BCR molecules can be strongly hindered by the signal from the plasma membrane-bound pool of the same molecules. To tackle this issue, we adapted the Specific Hybridization Internalization Probe (SHIP) assay, initially designed for flow cytometry studies, for the study of BCR internalization using microscopy. This assay utilizes a single-stranded DNA (ssDNA) fluorescence internalization probe (FIP) paired with a complementary ssDNA quenching probe that “turns off” the signal from the (extracellular) surface-bound BCRs, greatly facilitating the unambiguous identification of internalized (intracellular) receptors. Moreover, the assay is versatile and adaptable to a range of imaging modalities, including live-cell imaging and super-resolution microscopy. SHIP proves to be a valuable tool in the study of intracellular processes, offering enhanced imaging precision for the detection of internalized BCRs.