Alexa fluor 488 conjugated anti mouse or anti rabbit igg

Semithin (about 0.1 μm) ultramicrotomic sections were obtained from the resin blocks and used for immunofluorescence confocal and phase contrast microscopy. After washing with PBS, sections were incubated overnight with primary and then with secondary antibodies as described [23 (link)]. Primary antibodies against 19S, 20S, and polyubiquitinated proteins are the same used for electron microscopy. Secondary antibodies were Alexa Fluor 488-conjugated anti-mouse or anti-rabbit IgG (Life technologies, Paisley, UK). Specimens were analyzed by a TCS SP5II confocal laser scanning microscope equipped with PL APO 40x/1.25 NA and 63/1.40 NA oil-immersion objectives (Leica, Heidelberg, Germany).

HEK293A cells (2–4×10⁵ cells per 35 mm dish) were cultured on coverslips for 17 hrs, transfected with expression vectors, and incubated for another 24 hrs at 5% CO₂ at 37°C. Cells were fixed with 10% formalin for 20 min at room temperature and permeabilized with methanol for 20 min at room temperature. For proteasome inhibition, transfected cells were incubated in the presence of 30 µM MG132 or DMSO for 4 hrs prior to fixation. For immunostaining of the nuclear matrix-associated proteins, cells were sequentially treated with 0.5% Triton X100, 25 U/ml RNase-free DNase (RQ1, Promega), 0.25 M ammonium sulfate and 2M NaCl according to the method described [54] (link), [69] (link). Fixed cells were treated with PBS-Tween 20 containing 1% BSA (Sigma-Aldrich) for 1 hr at room temperature, incubated with primary antibodies to Myc (Cell Signaling; 1∶300), necdin (NC243;1∶1000) and NuMA (Ab-1, Oncogene;1∶100) overnight at 4°C, and with Alexa Fluor 488-conjugated anti-mouse (or anti-rabbit) IgG (Life Technologies;1∶1000) and cyanine 3-conjugated anti-rabbit (or anti-mouse) IgG (Jackson ImmunoResearch, 1∶500). Chromosomal DNA was stained with 3.3 µM Hoechst 33342 (Sigma-Aldrich). Stained cells were observed with a fluorescence microscope (BX-50-34-FLAD1, Olympus), and images were captured with a charge-coupled device camera system (DP-70; Olympus).