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2026-02
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Journal: STAR protocols
Article Title: Protocol for measuring interorganelle contact sites in primary cells using a modified proximity ligation assay.
doi: 10.1016/j.xpro.2024.102915
Figure Lengend Snippet: Figure 2. Images of PLA foci in transfected cells (A) Control PLA using IgG mouse antibody and TOMM20 (mitochondria). The PLA image demonstrates the specificity of the assay. (B) PLA for TOMM20 (mitochondria) and calnexin (endoplasmic reticulum). Boxed regions are enlarged in the bottom panels. White arrowheads show non-specific foci. Scale bar: 10 mm for the actual image and 5 mm for zoomed images. (C) Percentage of non-specific PLA foci (Calnexin-TOMM20, CLIMP63-TOMM20) in primary human fibroblast. Each point represents an individual cell. Bars show the average G SD.
Article Snippet: 1 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Mouse IgG isotype control 1:200 Invitrogen 08-6599 Fluorescent-tagged secondary antibody 1:500 Jackson ImmunoResearch 715-605-150; RRID:AB_2340862 (Anti-mouse 647), 711-165-152; RRID:AB_2307443 (Anti-Rabbit cy3) Chemicals, peptides, and recombinant proteins Dulbecco’s modified Eagle’s medium Wisent 319-005-CL Trypsin with EDTA Wisent 325-042-CL Penicillin-Streptomycin Wisent SV30010 Fetal bovine serum Corning 35-077-CV L-glutamine solution Wisent 609-065 EL 1X phosphate-buffered saline, sterile Wisent 311-425-CL Paraformaldehyde Sigma-Aldrich P6148 Bovine serum albumin Sigma-Aldrich A7906 Triton X-100 Sigma-Aldrich 9002-93-1 Epredia Immu-Mount Fisher Scientific 9990412 DAPI (40,6-diamidino-2-phenylindole, dihydrochloride) Thermo Fisher Scientific D1306 Critical commercial assays Duolink in situ green kit mouse/rabbit Sigma-Aldrich DUO92014 Duolink in situ PLA probe anti-rabbit PLUS Sigma-Aldrich DUO92002 Duolink in situ PLA probe anti-mouse MINUS Sigma-Aldrich DUO92004 Duolink in situ wash buffers, fluorescence Sigma-Aldrich DUO82049 Neon Transfection System 10 mL Kit Thermo Fisher Scientific MPK1025 Experimental models: cell lines Primary human fibroblasts (controls and DRP1 mutants) Skin biopsies (Research Ethics Board of the Children’s Hospital of Eastern Ontario (DRP1 mutants))
Techniques: Transfection, Control