Usp13

Immunohistochemistry staining was performed on 4-mm sections of FFPE lung specimens from 13 IPF patients and five normal controls. Paraffinized sections were deparaffinized in xylene and rehydrated through a series of alcohol to water. Slides were stained with hematoxylin and eosin (H&E) and viewed under a light microscope. For immunohistochemistry, the tissue sections were deparaffinized and rehydrated as described above. After a microwave treatment for 20 min in ethylenediaminetetraacetic acid (EDTA) buffer and subsequent cooling, the endogenous peroxidase activity was blocked by 0.3 % hydrogen peroxide in methanol for 15 min. After three washes followed by blocking in 5 % skim milk in phosphate-buffered saline (PBS) for 30 min, sections were incubated with antibodies against PTEN (clone 6H2.1, Cascade Bioscience, Winchester, MA, USA), USP13 (Abcam, Cambridge, MA, USA), and α-SMA (R&D, Minneapolis, MN, USA) overnight at 4 °C. Sections were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized using a 3, 3'-diaminobenzidine (DAB) kit (Maixin Biotech, Fuzhou, China). All slides were scanned using a Aperio ScanScope® AT (Leica, Nussloch, Germany), and staining of USP13 was analyzed with Aperio software.