Tcs spe rgbv confocal microscope

Two-day-old flies were anesthetized using FlyNap (Carolina), and heads and abdomens were removed. Thoraces were fixed overnight in 4% formaldehyde at 4 °C and rinsed in 1× Phosphate Buffered Saline (PBS, pH 7.4: 5.60 mM Na₂HPO₄, 1.06 mM KH₂PO₄, 154.0 mM NaCl) the next day (3 × 15 min, room temperature). Specimens were laid supine on a glass slide and snap-frozen in liquid nitrogen for 10 s. Frozen thoraces were immediately bisected down the mid-sagittal plane using a razor blade. Hemithoraces were stained with mouse anti-α-actinin antibody (1:50 in 1× PBS-T: PBS with 0.1% Triton-X 100) overnight at 4 °C. Samples were rinsed in 1× PBS the next day (3 × 15 min, room temperature), before being incubated with a mixture of secondary antibody (1:10,000 Alexa-488 goat anti-mouse, Invitrogen) and Alexa-568 phalloidin (1:50 in 1× PBS-T, Invitrogen) for 2 h at room temperature. Samples were rinsed in 1× PBS (3 × 5 min, room temperature) before being mounted on a glass slide with Vectashield (Vector Laboratories) and viewed using a 10× air lens to assess gross muscle morphology, and a 100× oil immersion lens (1.25 NA) to assess myofibrillar morphology on a Leica TCS SPE RGBV confocal microscope (Leica Microsystems, Buffalo Grove, IL, USA).

Patient myocardial samples were formalin-fixed, paraffin-embedded, cut at a 5μm thickness and mounted on clear, plus microscope slides. Slides were deparaffinized, rehydrated, underwent antigen retrieval, then blocked at room temperature for one hour as previously described [35 (link)]. Slides were incubated overnight at 4°C with mouse anti-N-cadherin (Santa Cruz, sc-59987; 1:500) or rabbit anti-Plectin (Cell Signalling, cs-D6A11; 1:400). The following day, slides were washed and incubated with secondary antibodies (donkey anti-mouse Alexa Fluor-647 [Invitrogen, A31571; 1:500] and goat anti-rabbit Alexa Fluor-488 [Invitrogen, A11070; 1:500]), washed and cover-slipped with mounting media (Fluoroshield with DAPI, Sigma F6057). Immunoreactive signal was visualized using a Leica TCS SPE RGBV confocal microscope (Leica Microsystems) at 40X magnification. Slides were coded and analysed in a blinded fashion by three independent observers.

Fluorescent microscopy of hemi-thoraces was performed^{36 (link),37 (link)}. Briefly, flies were anesthetized and their heads and abdomens were then removed. Thoraces were fixed overnight in 4% paraformaldehyde at 4 °C and rinsed in 1× phosphate buffered saline (PBS) the following day. The specimens were arranged on a glass slide, snap frozen in liquid nitrogen and bisected down the midsagittal plane using a razor blade. IFMs were stained with Alexa-Fluor 568 Phalloidin (1:100 in PBS with 0.1% Triton-X (PBST)) overnight at 4 °C, rinsed with PBS and visualized using EVOS® FL Cell Imaging System (Life Technologies) at ×4 magnification. For whole-mount imaging of IFM myofibrils, flies were prepared and thoraces bisected as described above. Hemi-thoraces were stained with Alexa-Fluor 568 Phalloidin (1:100 in PBST) overnight at 4 °C. Samples were rinsed in PBS, mounted with Vectashield (Vector Laboratories) and visualized using a Leica TCS SPE RGBV confocal microscope (Leica Microsystems) at 100x magnification.