Adamts12

Mouse tissue processing and immunohistostaining were performed by the Pathology Core Laboratory at Tulane University Health Sciences Center (http://medicine.tulane.edu/departments/pathologylaboratorymedicine/research/histology-laboratory) as described previously.19 (link), 20 (link) Heat-induced epitope retrieval was performed on tissue sections by using Rodent Decloaker solution (BioCare Medical, Concord, CA; RD913) and cooked in an oster steamer for 40 minutes. Sections were blocked by using Rodent Block M (BioCare Medical; RBM961), followed by incubation with the following antibodies: ADAMTS12 (Abcam; cat 203012, lot GR2390174), ITGA2 (Abcam; cat 133557, lot GR19622312), ST8SIA5 (Abcam; cat 184777, lot GR2231323), and Ki67 (1:100, 45 minutes; BioCare Medical; CRM325). After washing, tissue sections were incubated with Rabbit-on-Rodent HRP-Polymer secondary (BioCare Medical; RMR622); sections were then washed and treated with Betazoid DAB chromogen (Biocare Medical; BDB2004), followed by counterstaining with Cat hematoxylin (Biocare Medical; CATHEM). Slides were dried in the oven, placed in xylene, and coverslipped (Acrymount; StatLab, McKinney, TX; SL804). Images were obtained by using the slide scanner Aperio CS2 (Leica, Wetzlar, Germany) and Image Scope software.

Experimental cells were used for protein extraction, and immunoblot was performed as described previously.14 (link), 18 (link) The following specific antibodies against proteins were used: FOXO3 (Cell Signaling Technology, Danvers, MA; cat 2497, lot 8), ADAM metallopeptidase with thrombospondin type 1 motif 12 (ADAMTS12) (Abcam, Cambridge, United Kingdom; cat 203012, lot GR2390174; Thermo Scientific, Waltham, MA; cat PA568084, lot SK2479822E), ITGA2 (Abcam; cat 133557, lot GR19622312), ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 5 (ST8SIA5) (Abcam; cat 184777, lot GR2231323), and β-actin (Cell Signaling Technology; cat 3700, lot 8). Proteins were visualized with IRDye-conjugated secondary antibodies (LI-COR Biotechnology, Lincoln, NE; goat anti-mouse, cat 92532210, lot C6072602; goat anti-rabbit, cat 92568071, lot 92632210) using the Odyssey infrared imaging system (LI-COR Biotechnology).