Adi sab 100

Tissue samples isolated from an orthotopic xenograft model were used for histological analysis. Tissue samples were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5-µm sections. Paraffin-embedded tissue was deparaffinized, rehydrated, and subjected to antigen retrieval. The slides were blocked and incubated with primary antibodies against AGR2 (1: 500, ab76473), CD31 (1: 200, ab76533), cleaved caspase-3 (1: 500, Asp175, 9664), and PCNA (1: 5000, D3H8P, 13110) overnight at 4 °C, followed by incubation with secondary antibodies, namely, goat anti-rabbit IgG-horseradish peroxidase (HRP 1:500; ADI-SAB-100; Enzo Life Sciences, Farmingdale, NY, USA) or goat anti-mouse IgG-HRP (1:500; ADI-SAB-100; Enzo Life Sciences), Alexa Flour 488 goat anti-rabbit IgG (A11008, Invitrogen), and Alexa Flour 647 goat anti-rabbit IgG (A32733, Invitrogen), for 1 h at room temperature. The primary tumor and major organ sections were stained with H&E. Images were visualized using a Lion Heart FX automated microscope (Biotek, Winooski, VT, USA).

Treated and untreated cells were harvested after washing twice with ice-cold PBS. Cells were then lysed in Pro-prep (17081, iNtRON Biotechnology). Total cellular protein concentrations were quantified using a BCA assay kit (Thermo Fisher Scientific). Load samples containing equal amounts of protein in the SDS-PAGE wells were electrophoresed as previously reported [61 (link)]. The membranes were incubated with primary antibodies against AGR2 (ab76473), LC3B (ab48394), VEGF (A-20, sc-152), beta actin (sc-47778), and GAPDH (sc-32233) overnight at 4 °C, followed by incubation with secondary antibodies, goat anti-rabbit IgG-horseradish peroxidase (HRP 1:5000; ADI-SAB-100; Enzo Life Sciences, Farmingdale, NY, USA), or goat anti-mouse IgG-HRP (1:5000; ADI-SAB-100; Enzo Life Sciences) for 1 h at room temperature. Protein signals were developed using an Immobilon Crescendo Western HRP substrate (WBLUR0500) and were exposed on auto-radiography films.

Cell lysates were prepared to detect the protein expression of cleaved PARP1. Equal amounts of protein (25 μg) were boiled with Laemmli sample buffer (Bio-Rad, USA) for 5 min and electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels. Separated proteins were then transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore, USA) and blocked with 5% skim milk (Millipore) in Tris-buffered saline (TBS) containing 0.01% Tween-20 (TBST) for 30 min. The membranes were washed three times for 30 min in TBST and incubated with PARP1 (1:1,000 dilution, v/v, Cat#9542, Cell Signaling Technology, USA) and β-actin (1:2,000 dilution, v/v, Cat#sc-47778, Santa Cruz Biotechnology, USA) antibody in TBST buffer at 4°C overnight. The membranes were washed once every 10 min for 1 h in TBST and then incubated with secondary anti-rabbit (1:2,000 dilution, v/v, Cat#ADI-SAB-300, Enzo Life Sciences, USA) and anti-mouse (1:2,000 dilution, v/v, Cat#ADI-SAB-100, Enzo Life Sciences) HRP-conjugated antibodies for 1 h in TBST at room temperature. Primary and secondary antibodies were diluted with TBST and washed once every 10 min for 1 h. For visualization of the blots, the membranes were developed using D-Plus ECL Pico solution (Dongin Biotech, Korea).