Anti tag antibody

Analysis by immunohistochemistry was performed according to protocols described previously^{25 (link)}. Briefly, 3 μm serial sections were prepared from paraffin-embedded materials and subjected to hematoxylin–eosin (H&E) staining and immunohistochemistry using antibodies, including an anti-CK-19 antibody (The Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), an anti-TAg antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and an anti-Smad2/3 antibody (Cell Signaling Technology). For visualization of the immune complexes, the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) was used.

To estimate the occurrence of metastasis at the time of death or culling in TRAMP^MIC+/+ and TRAMP^MIC-/- mice, examined a different cohort of TRAMP^MIC+/+ (n = 63) and TRAMP^MIC-/- (n = 63). For comparison, we also examined a similar number of MIC-1/GDF15 overexpressing TRAMP^fmsmic-1 mice (n = 63), whose PCa was known to be associated with increased metastases [43 (link)]. Mice were looked after and euthanized using the same criteria as mentioned above in the survival study. At the necropsy pelvic lymph nodes, kidney, and liver tumors (if present) were harvested and fixed in 10% neutral buffered formalin. Lungs were excised, weighed and fixed in Bouin’s fixative (Sigma-Aldrich) to visualize and count lung tumor colonies. Metastatic lesions on all the organs were counted under a dissecting microscope. Some of the lesions were confirmed by H&E staining and further by immunostaining of frozen tissue sections with anti Tag antibody (Santa Cruz) to confirm the prostatic origin of the tumor. The number of mice having distant organ metastasis was compared in all the three mouse lines.