Donkey anti goat igg

Protein extracts from renal tissues were prepared by homogenization with glass tissue grinders (Kimble Chase, Rockwood, TN) in 1X cell lysis buffer (Cell Signaling, Danvers, MA). Samples were mixed with 1X Laemmli sample buffer, boiled, separated on a SDS-PAGE gel, and then transferred to PVDF membranes. Membranes were blocked with 5% nonfat dry milk, and then incubated overnight at 4° C with primary antibodies. Membranes were washed with TBST buffer (50 mM Tris, pH 7.4, 0.15 NaCl, and 0.05% Tween 20) and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Protein bands were visualized using the Clarity Western ECL kit (Bio-Rad, Hercules, CA) in a ChemiDoc MP system (Bio-Rad). The primary antibodies used were: monoclonal mouse-anti-β-actin (GenScript, Piscataway, NJ), polyclonal goat-anti-mouse ICAM-1 (R&D Systems, Minneapolis, MN), polyclonal rabbit-anti-rat ERK1/2 and polyclonal rabbit-anti-human phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling), polyclonal sheep-anti-mouse PC (Haematologic Technologies, Essex Junction, VT), and polyclonal rabbit-anti-mouse PAI-1 (ABCAM). The HRP-conjugated secondary antibodies used were: horse-anti-mouse IgG and goat-anti-rabbit IgG (Cell Signaling), donkey-anti-goat IgG (Jackson ImmunoResearch), and donkey-anti-sheep IgG (AbD Serotec, Raleigh, NC).

The maxilla was fixed overnight at 4°C in 4% paraformaldehyde/PBS solution, and then washed for 1 week in EDTA 0.5 M/PBS that was changed every other day. The tissue was cryopreserved in 30% sucrose (overnight at 4°C), embedded in OCT, and cryosectioned into 10 µm-thick sections. The cross sections, as well as the separated epithelium, were washed three times in PBS, blocked in a blocking buffer (5% FCS, 0.1% Triton X-100 in PBS) for 1 h at room temperature, and incubated with primary antibodies: goat anti-GAS6 (clone sc-1935, Santa Cruz Biotechnology), rabbit anti-PROS1 (clone 2428718, Millipore), rat anti-CD31 (clone 550274, BioLegend), goat anti-AXL (clone sc-1096, Santa Cruz Biotechnology), and rabbit-Anti-LYVE1 (clone-ab14917, Abcam) overnight at 4°C. Following three washing steps in PBS, the samples were incubated with a secondary antibody: donkey anti-goat IgG, donkey anti-rat IgG, or donkey anti-rabbit IgG (Jackson ImmunoResearch) diluted 1:100 in blocking buffer for 1 h at RT, washed three times, stained with hoechst, and mounted. As a negative staining control, primary antibody was omitted and replaced by blocking buffer. Signals were visualized and digital images were obtained using an Olympus BX51 fluorescent microscope mounted with a DP72 (Olympus) camera.

Prostate cancer tissue was used as a positive control in western blot analysis of purified CTRP3 protein. The specimen was collected from a prostate cancer patient confirmed by pathology. This study was ratified by the ethics committees of Central Hospital of Longgang District. The piece of tissue was crushed in liquid nitrogen, and then used to extract protein. Total cellular proteins were prepared from treated or untreated cells by lysing cells in lysis buffer (CelLytic, Sigma-Aldrich). Lysates were resolved on 10% or 12.5% polyacrylamide gels. Proteins were transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA), which was blocked with 5% milk containing TBS-T buffer (0.05% Tween-20) and incubated with primary antibodies at 4°C overnight. After washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG, donkey anti-goat IgG, or goat anti-mouse IgG antibodies (Jackson, USA). Immunoreactive bands were visualized with the Western Breeze chromogenic detection system (Invitrogen).

The cells were lysed with an appropriate volume of T-PER^TM Tissue Protein Extraction Reagent (#78510, Thermo Fisher Scientific) containing 1 × Halt^TM Protease and Phosphatase Inhibitor Cocktail (#78445, Thermo Fisher Scientific). The protein concentration was determined using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Ten micrograms of each protein sample was electrophoresed on 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto a nitrocellulose membrane. Western blot analysis was performed using the following primary antibodies and dilutions: mouse anti-NP (HB-65, ATCC, 1:50), mouse anti-non-structural protein 1 (NS1) (#sc-130568, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:1000), mouse anti-β-actin (Thermo Fisher Scientific, 1:3000), goat anti-IFIT1 (#PA5-848, Invitrogen, 1:1000), and mouse anti-IFIT2 (#sc-390724, Santa Cruz, 1:1000). The membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA, 1:2000), or donkey anti-goat IgG (Jackson ImmunoResearch, 1:2000) at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific).