Goat anti mouse alexa 555 secondary antibodies

Adult abdominal and larval muscles were dissected in saline and fixed with 4% paraformaldehyde for 15 min or Bouin's solution for 10 min. The dissected muscles were washed with 2% PBS-Triton X-100 and incubated with rabbit polyclonal antibody Ref(2)p/p62 (rabbit pAb, Abcam, ab178440) at a 1:500 dilution, TDP-43 rabbit polyclonal antibody (pAb, Proteintech, 10782-2-Ap) at a 1:500 dilution or Lamin C mouse monoclonal antibody (mAb, Developmental Studies Hybridoma Bank, LC28.26) at a 1:100 dilution overnight at 4°C on a nutator. Subsequently, the muscles were washed three times with 2% PBS-Triton X-100 and incubated overnight at 4°C with either goat anti-rabbit-Cy5 or goat anti-mouse-Alexa 555 secondary antibodies (Invitrogen) at a 1:500 dilution. Samples were mounted on a glass slide with Vectashield antifade mounting medium and imaged using a Leica DMi8 microscope with a 100× objective (1.4NA) and THUNDER imager.

Human NP cells were plated on a glass-bottom confocal dish and exposed to MCM for 48 h. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature, blocked with 5% bovine serum albumin (BSA; Millipore) in PBS, and then incubated with the primary antibodies overnight at 4 °C in 5% BSA. Anti-NF-κB p65 mouse monoclonal antibody (Santa Cruz) was used to detect the NF-κB p65 protein. Goat anti-mouse Alexa 555 secondary antibodies (Invitrogen) and 5% BSA were used for the secondary incubation in PBS for 1 h at room temperature. After washing in PBS, the plate was counterstained with 4′,6-diamidino-2-phenylindol (DAPI, Invitrogen). Images were acquired using the EVOS FL Auto cell imaging system (Thermo Fisher Scientific Inc., USA).