Ab103314

All tissues were fixed in 10% formalin, routinely dehydrated, dipped in wax, embedded in paraffin, and sliced to a thickness of 4 μm. Phosphate-buffered saline was used instead of primary antibody as a negative control. The primary antibody was PYCR1 (polyclonal rabbit antibody, ab103314; Abcam Inc, concentration: 1:100), dewaxing using xylene, dephenylation using ethanol, 3% dehydrogenation of endogenous peroxidase by hydrogen peroxide, repaired by high pressure of citric acid, and overnight refrigeration at 4°C. The secondary antibody (goat anti-rabbit antibody, SP-9000; Beijing Zhongshan Jinqiao Biotechnology Co, Ltd, Beijing, China) was incubated in a 37% water bath for 25 minutes, followed by DAB mean (3,3′-diaminobenzidine, DAB) coloration, hematoxylin staining, and sealing. PYCR1 protein expression was shown by brown staining mainly in the cytoplasm. PYCR1 expression was evaluated by 2 independent pathologists.
The expression of PYCR1 was measured by staining index. Scoring was based on the following criteria:
Staining index = positive staining cells + cell staining intensity, score (0–12). A staining index of >3 suggests a high expression, a staining index of ≦ 3 for low expression.^{[9 (link),11 (link)]}

Total protein was extracted from tissue lysate and the protein concentration was measured. An acrylamide gel was prepared at 10% for separation and 5% for loading, 40 μg of protein per well was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred to a polyvinylidene difluoride membrane, blocked, and exposed to rabbit anti-goat PYCR1 polyclonal antibody (ab103314; Abcam Inc, Cambridge, UK) overnight at 4°C. The secondary antibody was incubated for 2 hours at room temperature. β-actin was used as an internal reference. The relative expression of PYCR1 protein was calculated by using a biological imaging system. Each experiment was repeated 3 times.