Rabbit anti mta3 antibody

Placenta samples were fixed in neutral-buffered formalin for 3 days, processed, embedded in paraffin, and sectioned (5 μM-thick) for immunohistochemistry. Sections were blocked with 2% goat serum and 0.2% fish skin gelatin (Sigma) in 1X PBS for an hour at room temperature, then incubated with primary antibodies (rabbit anti-MTA3 antibody, mouse anti-hCG antibody (clone 5H4-E2), mouse anti- HLAG antibody (clone 4H84), all from Abcam; mouse anti-Ki67 antibody from Dako; or nonspecific mouse or rabbit IgG as negative controls) at 4 °C overnight. After three washes with 1X PBS with 0.1% Tween-20, sections were incubated with secondary antibodies (Alexa Fluor 488- or 568-conjugated goat anti-mouse or anti-rabbit, from Invitrogen) and DAPI for one hour at room temperature, then mounted with anti-fade mounting solution (Invitrogen) and examined using a Leica fluorescence microscope. Adjacent sections were also stained using hematoxylin and eosin, according to standard protocols, and examined using an Olympus light microscope.

Total protein was obtained following cell lysis in 1% TritonX-100 and 0.5% SDS in 1X Tris-buffered saline (TBS) and sonication. Protein concentration was quantified by Pierce BCA protein assay (Thermo Scientific). 25–30 μg of protein were separated on a 10% SDS-PAGE gel. After transfer to a polyvinylidene fluoride (PVDF) membrane (BioRad), western blotting was performed as previously described [10 (link)] using rabbit anti-MTA3 antibody (Abcam) and mouse anti-β-actin (Sigma).