Ab116082

For the IHC analysis, fresh specimens were cryopreserved and routinely processed into frozen sections. Then, 4-μm-thick sections were prepared, and immunohistochemical staining with a streptavidin-biotin immunoperoxidase assay was performed using rabbit antibodies against HSPA12B (1:400 dilution, ab116082; Abcam). The slides were imaged under a light microscope (Leica, Germany) at 100× magnification. Brown staining in the cells was considered a positive signal.

A total protein extraction kit (KeyGen Biotech, Nanjing, China) was used to extract total proteins. The procedures were followed according to the kit manual. Cell protein lysates were separated by 10% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany) using a standard wet transfer apparatus. Then, the membranes were blocked in 5% non-fat milk in Tris-buffered saline with 0.05% Tween 20 (TBST) at room temperature for 2 hr, incubated overnight with the primary anti-HSPA12B (1:1,000 dilution, ab116082; Abcam), anti-Bcl2 (1:1,000, ab32124; Abcam), anti-Bax (1:1,000, ab77566; Abcam), anti-cleaved Caspase-3 (1:1,000, ab2302; Abcam), or anti-GAPDH antibody (1:1,000 dilution, AF0006; Beyotime); anti-GAPDH was used as an internal control. The membrane was washed with TBST three times for 5 min per wash. Then, the PVDF membrane was incubated in blocking buffer containing the diluted secondary antibody at room temperature for 2 hr. Finally, protein bands were detected on FluorChem FC2 Imaging System (Alpha Innotech, San Leandro, CA, USA).