Hrp conjugated rabbit secondary antibody

The overall protein concentration in sera was measured using the Bradford Protein Assay (Bio-Rad, Hercules, CA, USA), as described previously [39 (link)]. Equal amounts of total protein (20μg) were resolved on a 10 well SDS-PAGE gel. Precast gels (NuPAGE Novex 4–12% Bis-Tris Protein Gels) were obtained from Invitrogen/ThermoFisher Scientific (Grand Island, NY). Western blotting was performed as described previously [39 (link)]. A 1:1000 dilution of polyclonal rabbit DβH primary antibody was prepared in 5% BSA in phosphate buffered saline-tween 20 (PBS-TT) to probe the membrane at 4°C overnight (polyclonal DβH antibody was obtained from Cell Signaling Technology, Danvers, MA). The blots were then developed with 1:5000 dilution of an anti-rabbit secondary antibody prepared in 5% dry milk in PBS-TT (HRP-conjugated rabbit secondary antibody was obtained from Cell Signaling Technology, Danvers, MA). An enhanced chemiluminescent (ECL) reagent was employed to visualize the immunoblot signal (Pierce, ThermoFisher Scientific, Grand Island, NY). Transferrin was used as a loading control (monoclonal transferrin primary antibody was obtained from Abcam, Cambridge, MA). ImageJ software [40 (link)] was employed to quantify the intensity of each band. The DβH band intensities were normalized to transferrin and the expression levels of DβH in different subjects were compared.

After fixation and routine dehydration, all tumor samples were embedded in paraffin and cut into 2-μm thick sections. The xenografted specimens for histological analysis were stained with hematoxylin and eosin (HE) in order to observe the general tissue morphology under the DM4000B microscope (Leica, Germany). For immunohistochemistry, sections were deparaffinized using xylenes for 10 min each and hydrated using a graded alcohol series (100 to 75%) for 5 min each. Antigen retrieval was performed by heating the sections in citrate buffer for 2 min in a pressure cooker. The endogenous peroxidase activity was inactivated using 0.3% hydrogen peroxide for 10 min at RT in the dark. Afterwards, sections were incubated with a 1:8000 dilution of anti-PCNA antibody and 1:800 dilution of anti-P62 antibody (Cell Signaling Technology, USA) overnight in a moist chamber at 4 °C. The next day, the sections were washed and incubated with an HRP-conjugated rabbit secondary antibody (Cell Signaling Technology, USA) for 30 min at RT. The PCNA and p62 signals were detected by using DAB substrate (brown). All tumor sections were counterstained with haematoxylin for 1 min, dehydrated, dried, and mounted using Permount TM Mounting Medium. The images were captured using the LEICA DM4000 B LED microscope (Leica, Germany).