In Situ PLA for FKBP12/RyR Stability in Myotubes

In situ PLA was used for evaluation of FKBP12/RyR stability in human myotubes under nitro-oxidative stress, as previously described [19 (link)]. Briefly, immortalized human myoblasts (Myology Institute, Paris, France) were differentiated into myotubes and nitro-oxidative stress was induced by addition of the peroxynitrite donor 3-morpholinosydnonimine (SIN1, 5 mM, SCBT, Heidelberg, Germany) for 30 min. Myotubes were fixed with 4% paraformaldehyde (Aname, Madrid, Spain) and incubated overnight with the following primary antibodies: anti-RyR mouse mAb (1:200, Thermo Scientific, Waltham, MA, USA), and anti-FKBP12 rabbit pAb (1:100, Novus Biologicals, Centennial, CO, USA). The Duolink in situ PLA Orange assay kit was used with corresponding conjugated secondary antibodies (Sigma). Samples were counterstained with FITC-conjugated Myosin Heavy Chain-CFS mAb (MyHC, 1:50, R&D, Minneapolis, MN, USA) and mounted with ProLong Gold antifade reagent (Life Technologies, Eugene, OR, USA). Image quantification was performed with ImageJ (NIH). For each image, the total number of spots was normalized to the MyHC area. At least 3 images per condition were analysed with an average of 8–9 myotubes per image. Statistical significance was determined using One-Way ANOVA followed by Dunett’s multiple comparisons test. p-values < 0.05 were considered statistically significant.