Rabbit anti c ebpβ sc 150

Whole cell lysate were prepared in IPH buffer (50mM Tris pH 7.5, 150mM NaCl, 0.5% NP-40, 5mM EDTA, 1mM DTT and 1X protease inhibitor cocktail) and briefly sonicated on ice. To assess expression of myoblast and myotube markers, the following antibodies were used: mouse anti-MyoD (SC-32758, 1/500), mouse anti-myogenin (SC-12732, 1/500), rabbit anti-C/EBPβ (SC-150, 1/500) all from Santa Cruz Biotechnology, mouse anti-Pax7 (pax7, 1/200) and mouse myosin heavy chain (MF20, 1/100) both from DSHB. Rabbit anti-Cyclophilin B (Abcam, 1/10,000) and mouse anti-β-actin (Sigma, 1/10,000) were used as loading controls. Secondary HRP-conjugated antibodies used were donkey anti-rabbit (NA934, 1/2,000) and sheep anti-mouse (NA931, 1/2,000) both from Amersham GE Healthcare Life Sciences. Chemiluminescence images were captured using the Luminescent Image Analyzer LAS-4000 (Fujifilm Life Science).

For immunocytochemistry, cells were washed, fixed in 2% paraformaldehyde, permeabilized with 0.5% Triton X-100 and blocked 1 hour in 5% normal donkey serum. Following washes, mouse anti-MyHC (MF20, 1/200), mouse anti-Pax7 (Pax7, 1/100) both from DSHB, or rabbit anti-MyHC (SC-20641, 1/100) primary antibody was added and incubated at 4°C. Secondary antibodies used were: Alexa488-conjugated donkey anti-rabbit (711-546-152, 1/500), biotin-conjugated donkey anti-mouse (715-066-150, 1/500), Cy3-conjugated streptavidin (016-160-084, 1/1,000) all from Jackson Immuno-Research. Nuclei were identified by DAPI staining.
Indirect immunofluorescence was performed on frozen sections of TA muscle from C57BL/6 mice. 8μm-thick sections were dehydrated 30min at 37°C, fixed in 4% paraformaldehyde and antigen-retrieval was perform by incubating sections at 92°C in citrate buffer (10mM citric acid, 0.05% Tween-20, pH 6.0) for 20min. Primary antibodies used were rabbit anti-C/EBPβ (SC-150, 1/200) from Santa Cruz Biotechnology, anti-Pax7 (pax7, 1/100) from DSHB. Secondary antibody labelling was done as above. For image acquisition, pictures were taken on a DMI3000B epifluorescence microscope (Leica) using infinity 3 (Lumenera) camera. For processing, individual pictures were level adjusted uniformly and pasted on a single color channel in Adobe Photoshop.