Mouse anti human nuclear antigen

The recombination assays were fixed and permeabilized as described above. Then blocked overnight at 4°C in 10% DS, 1% BSA, 1.8% IgG MOM and 0.1% PBST. Primary antibodies were incubated overnight at 4°C in 16% MOM and 0.1% PBST: Pna-Rhodamine (1:75), rabbit anti-K5 (1:2000), mouse anti-Tubb3 (1:200), rabbit anti-Ecadherin (1:100), rat anti-perlecan (1:100). Primary antibodies for human-mouse recombination incubated overnight at 4°C in 16% MOM and 0.1% PBST: mouse anti-Ki67 (1:100), goat anti-Kit (1:100), mouse anti-human nuclear antigen (1:100, Millipore), rabbit anti-Ecadherin (1:100), mouse anti-Tubb3 (1:200), rat anti-integrinβ1 (1:100, Gift of Dr. K.M. Yamada) and rabbit anti-K5 (1:2000). Secondary antibodies incubated for one hour at room temperature in 16% MOM protein and 0.1% PBST: 1:2000 Hoescht, anti-Rabbit cy3 and cy5 (1:200), anti-Mouse cy2 (1:100), anti-goat cy 5 (1:200), and anti-rat cy3 and cy2 (1:200).

Snap frozen tumor blocks were cryo-sectioned through the University of Michigan microscopy and imaging lab. Sections were fixed in 4% formaldehyde and permeabilized with 0.1% triton. Sections were stained with either mouse anti-human nuclear antigen (Millipore), anti-TP53 (abcam), anti-PAX8 (abcam), anti-human mitochondrial antibody (Life Technologies), anti-human CD34 (abcam) or anti-smooth muscle actin-Cy3 antibody (abcam) in 1:100 dilution. Of note, anti-human CD34 antibody used is predicted to detect mouse in addition to human CD34. Goat anti-mouse-Alexa 488 secondary antibody was then applied (Life Technologies). Images were obtained using an Olympus BX41 microscope. Paraffin imbedded tumor tissue was prepared, sectioned and stained through the University of Michigan Microscopy & Image Analysis core.