Fitc labeled goat anti mouse antibody

Using flow cytometry, primary rat hepatocytes in cell fibers were identified by detecting Asialoglycoprotein receptor-1 (ASGPR-1) based on previous report^{33 (link)}. First, the hydrogel shells of the cell fibers are dissolved using alginate lyase (Sigma Aldrich), and the cells of the core were dissociated using collagenase (Brightase, Nippi, Japan). These cells were resuspended in DMEM supplemented with 10% of FBS, incubated for at least 30 min, and then centrifuged at 400 g. The cells were treated for 30 min with a 100 µL solution containing a mouse-anti-human ASGPR1 antibody (Thermofisher, Waltham, MA) diluted 1:50. A fluorescein isothiocyanate (FITC)-labeled goat anti-mouse antibody (Thermofisher, Waltham, MA) was added to the solution at the concentration of 0.5 g mL⁻¹, and the cells in the solution was incubated for 30 min. The cells were washed 3 times with 1 mL fluorescence-activated cell sorter buffer (1% sodium azide and 2% fetal bovine serum in phosphate-buffered saline), resuspended in 500 µL fluorescence-activated cell sorter buffer, and analyzed by flow cytometry. Cell analysis was performed on a Becton–Dickinson FACSVerse flow cytometer (Becton–Dickinson Biosciences, San Jose, CA) and data acquisition and analysis was performed using Becton–Dickinson FACSuite software (Becton–Dickinson Biosciences, San Jose, CA).

The pET28a expression system was obtained from GE Healthcare; the pEGFP-N1 vector was obtained from Clontech; The DNA encoding for segment from 259 amino acids to 345 amino acids of glycoprotein D (AFB76672.1) was amplified by polymerase chain reaction with the primers as following: Sense primer: 5′-GAATTCATGGAGGAGTCGAAGGGC-3′ and anti-sense primer:5′-CTCGAGGATGGCTTCGAGGCTCG-3′, and the DNA fragment was cloning into the pEGFP-N1 vector for construction of the pEGFP-N1-gD, which was used to efficiently express green fluorescent protein (GFP) fused gD protein in 293T cell. Calf antiserum against BoHV-1 was from China Veterinary Culture Collection Center. A mouse anti-His monoclonal antibody (McAb), Alexa Fluor 555- conjugated anti-His antibody, FITC-labeled goat anti-mouse antibody and TRITC-labeled goat anti-mouse antibody were purchased from ThermoFisher Scientific (United States), and a FITC-labeled rabbit anti-bovine antibody was purchased from BioVision (United States). The antibodies against PARP-1, Bcl-2, Bid, caspase-3, caspase-8, caspase-9 and β-actin were purchased from ABclonal Biotech (China). The BoScFv-PE38 was labeled with horseradish peroxidase (HRP) by Sangon Biotech Co., Ltd. (China).