Goat anti rabbit igg

We prepared 4 μm thick sections for each group of tumor tissues. The sections were labeled using anti-Ang-1, anti-Tie-2, anti-SCF, and anti-Flt-3L primary antibodies (Abcam; at 1:200, 1:50, 1:200, 1:180, respectively) at 4°C overnight and then incubated with a secondary antibody (goat anti-rabbit IgG; Zhongshan Golden Bridge, Beijing, China) for 1 h at room temperature. Following DAB coloration, hematoxylin counterstaining, dehydration and clearing in xylene, the slides were mounted. Staining intensity and positive area were analyzed by the average density (Average Optical Density, AOD) using image J software (V1.8.0.172). AOD =Integral Optical Density (IOD)/Positive area.

Mouse polyclonal antibodies were raised against recombinant dZIP13-2 protein fragment (MTEEKMAKEGYKDPADSKLLRSGSADEENPQPKCVEIANCLLRRHGGQLPEGETSESCGGACDIEDVGKVCFLREQEQKSKERKEQPKRSGFSRWDAARAQKEEERKESIKQLE). Briefly, the cDNA fragments encoding the cytosolic side of this protein (dZIP13-2) were synthesized and cloned into pTwin1 (NEB) vector. The recombinant protein was expressed in E. coli and purified by chitin beads (NEB), and injected into mice for antibody generation (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China). The antibody was affinity purified and pre-absorbed before use. Anti-Fer2LCH was as described before (Tang and Zhou, 2013b (link)). Anti-tubulin rat monoclonal antibody (ab6160), anti-GM130 rabbit polyclonal antibody (ab30637), and anti-PDI mouse monoclonal antibody (ab2792) were obtained from Abcam (Cambridge, MA, USA). Secondary antibodies include HRP-conjugated goat anti-mouse IgG, goat anti-rabbit IgG and goat anti-rat IgG (Zhongshan Goldenbridge Biotechnology, Beijing, China). For Western blot analysis, fly samples were homogenized in the buffer containing 1% Triton X-100 plus 10% proteinase inhibitor cocktail (Sigma), centrifuged, separated on 10% SDS-PAGE, and transferred to nitrocellulose membranes (Millipore, Watford, UK). Signals were developed with ECL detection kit (Vigorous Biotechnology, Beijing, China).

Liver tissues were removed at sacrifice and then fixed in formalin, embedded in paraffin, and prepared for histology. The liver tissue slides were blocked with 10% goat serum for 30 min, followed by incubation for 1 h with anti‐F4/80 antibody (1:200; ab16911, Abcam, China) and anti‐pS6 antibody (1:200; 2211, CST, USA) at 4°C overnight. After incubation with a poly‐peroxidase‐conjugated goat anti‐rat IgG or goat anti‐rabbit IgG (Zhongshan Golden Bridge, China) at 37°C for 30 min, the slides were counterstained with 3,3′‐diaminobenzidine (DAB) and hematoxylin and eventually mounted with glycerin.

Western blotting was used to examine the expression of AKT, phosphorylated AKT (p-AKT), Nrf2, and HO-1. Total proteins (20 μg) from each sample were electrophoresed on a 12% SDS-polyacrylamide gradient gel and transferred to nitrocellulose membranes (Millipore). The membrane was blocked with 5% fat-free milk in rinse buffer for 30 min and incubated for 2 h with the following primary antibodies: AKT antibody (1 : 1000, Proteintech), p-AKT antibody (1 : 1000, Santa Cruz Biotechnology), Nrf-2 (1 : 1000 Proteintech), HO-1 (1 : 500 Proteintech), and anti-β-catenin (1 : 1000, Abcam). Next, they were incubated with an HRP-conjugated secondary antibody (goat anti-rabbit IgG 1 : 5000, Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd.) and visualized using the enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL, USA). β-Actin was used as a reference protein.

Paraffin sections of mouse aorta and human aorta were deparaffinized. The tissue array (Alenabio, China) used for immunohistochemistry of human tissue contained continuous tissue sections for von Kossa staining. The sections were incubated with an anti-CA1 polyclonal antibody (Cusabio, China) overnight at 4°C. The sections were then incubated with goat anti-rabbit IgG (Zhongshan Golden Bridge Biotechnology, China). Sections were treated with diaminobenzidine (DAB, Zhongshan Golden Bridge Biotechnology, China) and counterstained with hematoxylin.

Immunohistochemical specimens were fixed in 10% formaldehyde, which were then embedded in wax blocks. This study obtained anti-DDX56 antibody in Santa Cruz Biotechnology (5A7: sc-101,018, Santa Cruz, CA) and Ki-67 polyclonal antibody in Proteintech Group (No. 26,593-1-AP, ProteinTech, Wuhan, China). Following manufacturer’s instruction, the endogenous peroxidase blocking reagent (SP-9000, Zhongshan Golden Bridge, Beijing, China), goat serum (SP-9000, Zhongshan Golden Bridge), anti‐DDX56 antibody, Ki-67 polyclonal antibody, goat anti-mouse IgG (SP-9000, Zhongshan Golden Bridge), goat anti-rabbit IgG (SP-9000, Zhongshan Golden Bridge), HRP-labeled avidin working fluid (SP-9000, Zhongshan Golden Bridge) and Diaminobenzidine (DAB; ZLI-9018, Zhongshan Golden Bridge) were added in a regular sequence. Finally, the tissue sections were stained, and then a light microscope (Olympus Corporation, Tokyo, Japan) was employed for observation. Tumor histology was independently reviewed by an experienced pathologist. The DDX56 levels were rated as 0–3, indicating negative, low, moderate, and high intensities, respectively. The scores regarding the extent of staining were 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). In addition, a score of >4 was suggested as high.

Total protein extraction of NK cells, NK-92 cells lines and tumor tissues was performed using RIPA lysis buffer (Beijing Solarbio Science and Technology, Beijing, China). Protein samples were separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore) and blocked with tris buffered saline tween (TBST) buffer containing 5% non-fat milk at room temperature for 1 h. The membranes were incubated with primary antibodies rabbit anti-RUNX3 (1:1000; Cell Signaling Technology, Boston, MA, USA) and anti-NKp46 (1:1000; Bioworld Technology Inc. Minneapolis, MN, USA) at 4 °C overnight, followed by rinsed with TBST thrice, and then incubated with goat anti-rabbit IgG (Zhongshan Golden Bridge Biotechnology, Beijing, China) at room temperature for 1 h under a shaker. Protein bands were visualized using an enhanced chemiluminescence reagent (Beckman Coulter, Brea, CA, USA). Band intensities were standardized to β-actin (Sigma-Aldrich) as a loading control.