Alexa fluor 488 goat anti mouse igg

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.

After anesthetization with diethyl ether, mice were transcardially perfused with PBS followed by 4% paraformaldehyde (PFA). Brains were post-fixed in 4% PFA for 2–4 h. Free-floating coronal sections (40 μm) were prepared using a cryostat. The sections were incubated with 0.2% Triton-X-100 and 5% goat serum for 1 h, primary antibodies including polyclonal anti-Zif-268 antibody (SC-189, 1:5000; Santa Cruz) and anti-green fluorescent protein (GFP) primary antibody (A11120, 1:1000, Invitrogen) at 4 °C overnight, followed by incubation with secondary antibodies including goat anti-rabbit biotinylated antibody (A31553, 1:400; Life Technologies) and Alexa Fluor 488 goat anti-mouse IgG for 2 h, VECTASTAIN ABC Kit (Vector Laboratories) for 1.5 h, and TSA-Cyanine 3 (SAT704A001EA, 1:1000; Perkin–Elmer) for 1 h. The sections were mounted in PermaFluor (ThermoShandon, Pittsburgh, PA, United States). Nuclei were counterstained with Hoechst dye (1:1000; Invitrogen).