Optimizing In Situ Proximity Ligation Assays for Mitochondria, ER, or MERC Markers in Skeletal Muscle Tissue and Cells

Stephens, D. C.; Crabtree, A.; Beasley, H. K.; Garza-Lopez, E.; Neikirk, K.; Mungai, M.; Vang, L.; Vue, Z.; Vue, N.; Marshall, A. G.; Turner, K.; Shao, J.-q.; Murray, S. A.; Gaddy, J. A.; Wanjalla, C.; Davis, J.; Damo, S. M.; Hinton, A.

doi:10.1101/2023.05.20.541599

bioRxiv · 10.1101/2023.05.20.541599

Optimizing In Situ Proximity Ligation Assays for Mitochondria, ER, or MERC Markers in Skeletal Muscle Tissue and Cells

Stephens, D. C.·Crabtree, A.·Beasley, H. K.·Garza-Lopez, E.·Neikirk, K.·Mungai, M.·Vang, L.·Vue, Z.·Vue, N.·Marshall, A. G.·Turner, K.·Shao, J.-q.·Murray, S. A.·Gaddy, J. A.·Wanjalla, C.·Davis, J.·Damo, S. M.·Hinton, A.

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Abstract

Proximity ligation assays (PLA) use specific antibodies to detect endogenous protein-protein interactions. PLA is a highly useful biochemical technique that allows two proteins within close proximity to be visualized with fluorescent probes amplified by PCR. While this technique has gained prominence, the use of PLA in mouse skeletal muscle (SkM) is novel. In this article, we discuss how the PLA method can be used in SkM to study the protein-protein interactions within mitochondria-endoplasmic reticulum contact sites (MERCs). Tweetable AbstractProximity Ligation Assays can be used in skeletal muscle tissue and myoblasts to explore the protein-protein interactions involved in MERC sites. HighlightsO_LISkeletal muscle tissue and cells are plated on glass coverslips for evaluation by proximity ligation assay (PLA). C_LIO_LIFollowing fixation, cells are probed and stained for Mfn1, Mfn2, mitochondria, and ER and imaged using fluorescence confocal microscopy. C_LIO_LIThis method shows that PLA can be used in mouse SkM and is adaptable to other models. C_LIO_LIProtocol for detection of protein-protein interactions using PLA. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/541599v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1021ce7org.highwire.dtl.DTLVardef@1b78f8borg.highwire.dtl.DTLVardef@31030corg.highwire.dtl.DTLVardef@1bf0afa_HPS_FORMAT_FIGEXP M_FIG C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@169ba8corg.highwire.dtl.DTLVardef@566b30org.highwire.dtl.DTLVardef@197330dorg.highwire.dtl.DTLVardef@198ca7borg.highwire.dtl.DTLVardef@158b80_HPS_FORMAT_FIGEXP M_TBL C_TBL

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Stephens, D. C., Crabtree, A., Beasley, H. K., Garza-Lopez, E., Neikirk, K., Mungai, M., Vang, L., Vue, Z., Vue, N., Marshall, A. G., Turner, K., Shao, J.-q., Murray, S. A., Gaddy, J. A., Wanjalla, C., Davis, J., Damo, S. M., Hinton, A.. 2023-05-22. Optimizing In Situ Proximity Ligation Assays for Mitochondria, ER, or MERC Markers in Skeletal Muscle Tissue and Cells. https://doi.org/10.1101/2023.05.20.541599

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