Rhodamine phalloidin phalloidin 555

Immunostaining of myofiber preparations was performed as previously described (Horstick et al., 2013) . Briefly, myofiber preparations were fixed with 4% PFA, for 20 min, permeabilized with PBST (0.3% TritonX in PBS), blocked for 1 hour with PBSTB (5% BSA in PBT) and incubated overnight at 4 o C with primary antibodies. The following primary antibodies were used: mouse anti-DHPR (1:200; DHPRa1A; Abcam), mouse anti--Actinin (1:100; Sigma), mouse anti-RyR1 (1:100; 34C; DSHB), rabbit anti-Junctin (1:350; gift from Dulhunty lab), mouse anti-PI(3)P (1:100; Echelon Biosciences Inc.), mouse anti-PI(3,4)P2 (1:100; Echelon Biosciences Inc.), mouse anti-PI(4,5)P2 (1:100; Echelon Biosciences Inc.). Alexa Fluor-conjugated secondary antibodies were used at 1:1000 (Invitrogen). Rhodamine phalloidin (Phalloidin 555) was used to visualize filamentous actin (1:300, Molecular Probes). Preparations were mounted with ProLong Gold with DAPI (Invitrogen). Images were acquired with a Nikon Eclipse Ti laser scanning confocal using NIS Elements software (company, location) and only adjusted for brightness and contrast using Adobe Photoshop.