Goat anti rabbit igg h l alexa fluor 594 secondary antibody

A nuclear location of NOX2 in H9c2 cells was observed by confocal microscopy. In brief, H9c2 cells were fixed with 4% paraformaldehyde for 10 min. And after washing twice with PBS, using 0.5% Triton X-100, H9c2 cells were permeabilized. After washing twice again, using 1% Bull Serum Albumin (BSA) to block cells for 30 min at room temperature and then washing three times with PBS, the cells were incubated with primary antibodies against Nox2 (1 : 5000, Abcam) overnight at 4°C. The next day, the cells were washed three times with PBS and incubated with Goat Anti-Rabbit IgG (H&L) Alexa Fluor 594 secondary antibody (1 : 200, Abcam) for 2 h at room temperature. After washing twice with PBS, H9c2 cells were incubated with 4′, 6-diamidino-2-phenylindole (DAPI) to stain nuclei for 30 min without light. Finally, H9c2 cells were observed under confocal microscopy.

Muscle was dissected from 521ΔT D2BA/2J F5 mice bred to homozygosity along with the muscle from WT littermates. Muscle was flash frozen in liquid nitrogen. Total RNA was isolated from the tibialis anterior as described below, and analyzed for the expression of the Sgcg and 521ΔT transcripts using the following primer sets: F, 5′-ACTCACATAGAGAGGCCCGA-3′; R, 5′-CCATCAGGACACGCACAGAT-3′. Individual bands were gel extracted, purified using the Qiaex 2 Gel Extraction Kit (20021, Qiagen) and submitted for Sanger sequencing. Extracted PCR products were also subcloned into the TOPO PCR 2.1 vector described above, mini-prepped and submitted for Sanger sequencing. For detection of γ-sarcoglycan, 10 µm muscle sections were fixed for 2 min in ice-cold methanol. After fixation, muscle was rinsed with PBS, blocked in PBS plus 10% fetal bovine serum and 0.1% Triton X-100 (9002-93-1, Sigma-Aldrich) for 1 h at 4°C, then incubated with anti-γ-sarcoglycan antibody (1:250; McNally, 1996 (link)a). Sections were rinsed and incubated with goat anti-rabbit IgG H&L Alexa-Fluor-594 secondary antibody (150080, Abcam, 1:2500) and Hoechst 33342 (H3570, Thermo Fisher Scientific, 1:10,000) for 1 h at room temperature. Sections were fixed in Vectashield mounting media (H-1000, Vector Laboratories) and imaged on a Zeiss Observer microscope using Zen Pro software.