Phalloidin alexa568

Immunostaining was performed on 5 μm‐thick muscle cryosections, fixed in 4% PFA and incubated with PBS containing 2% FBS for 30 min or on fixed cells blocked with PBS 2% FBS 0.2% Triton. Cryosections or fixed cells were incubated for 1 h with primary antibodies (Table3). After three washes in PBS, immune complexes were detected by incubation with the appropriate Alexa Fluor‐conjugated secondary antibodies purchased from Life Technologies at RT for 45 min. Nuclei and actin filaments were stained with Hoechst and phalloidin‐Alexa568 (Interchim), respectively.

Myoblasts were fixed for 5 min with 4% formaldehyde, permeabilized with 0.5%Triton X100 and blocked with 5% bovine serum albumin (BSA) diluted in PBS. Myoblasts were stained with Phalloidin-Alexa 568 to label F-actin (Interchim, Montluçon, France). The following primary antibodies were used for immunostaining: anti-vinculin (Sigma-Aldrich, Saint Quentin-Fallavier, France), anti-non muscle myosin IIA (NM-2A) (Abcam, Paris, France), anti-lamin A/C (sc-6215, Santa Cruz Biotechnology, Santa Cruz, California, USA), anti-emerin (NCL-emerin Novocastra), and anti-SUN2^{11 (link)} (generously provided by D. Hodzic), anti-nesprin 1 (N1G-7C8 and MANNES1A), which recognize exons 84-85 and exons 143-146 of nesprin-1, respectively)^{54 (link)}, anti-nesprin-2 (MANNES2A)^{54 (link)}, and anti FHOD1 (ab73443, Abcam). Secondary antibodies (Life technologies, Saint-Aubin, France; 1/500) were: Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 568 goat anti-rabbit IgG, Alexa Fluor 488 donkey anti-mouse IgG, or Alexa Fluor 488 rabbit anti-goat IgG. The preparations were mounted on slides with fluorescent mounting medium containing DAPI (Vectashield, Vector Labs, Berlingame, California). Confocal images were taken with an Olympus FV 1000 (Olympus, Hamilton, Bermuda) and a Leica SP2 (Leica Microsystems, Wetzlar, Germany) microscopes.